Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The...

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Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins Yana K. Reshetnyak and Edward A. Burstein Institute of Theoretical and Experimental Biophysics, Russia Academy of Sciences, Pushchino, Moscow Region, Russia 142290 ABSTRACT The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores in proteins have been proposed in the model of discrete states (Burstein, E. A., N. S. Vedenkina, and M. N. Ivkova. 1973. Photochem. Photobiol. 18:263–279; Burstein, E. A. 1977a. Intrinsic Protein Luminescence (The Nature and Application). In Advances in Science and Technology (Itogi Nauki i Tekhniki), Biophysics Vol. 7. VINITI, Moscow [In Russian]; Burstein, E. A. 1983. Molecular Biology (Moscow) 17:455– 467 [In Russian; English translation]). It was assumed that the existence of the five most probable spectral classes of emitting tryptophan residues and differences among the classes were analyzed in terms of various combinations of specific and universal interactions of excited fluorophores with their environment. The develop- ment of stable algorithms of decomposition of tryptophan fluorescence spectra into log-normal components gave us an opportunity to apply two mathematically different algorithms, SImple fitting with Mean-Square criterion (SIMS) and PHase- plot-based REsolving with Quenchers (PHREQ) for the decomposition of a representative set of emission spectra of proteins. Here we present the results of decomposition of tryptophan emission spectra of .100 different proteins, some in various structural states (native and denatured, in complexes with ions or organic ligands, in various pH-induced conformations, etc.). Analysis of the histograms of occurrence of .300 spectral log-normal components with various maximum positions confirmed the statistical discreteness of several states of emitting tryptophan fluorophores in proteins. INTRODUCTION Tryptophan fluorescence is widely used to study the loca- tion, physical and dynamic properties of microenvironment of indole fluorophores, and the structural features and be- havior of the protein molecule as a whole (Burstein, 1976, 1977a, 1983; Lakowicz, 1983; Demchenko, 1986). Depend- ing on the environment of tryptophan residues in proteins, the maximum position (l m ) and quantum yield (q) of tryp- tophan fluorescence could vary widely, from 308 to 353 nm and from 0.4 to immeasurably low, respectively. In 1967 Konev put forward the hypothesis of the exis- tence of two main classes of tryptophan residues in proteins, which possess discrete values of fluorescent parameters l m and q (Konev, 1967; Volotovski and Konev, 1967). One of the classes included tryptophan fluorophores inside the pro- tein in a low-polar hydrophobic environment with a shorter- wavelength position of fluorescent maximum (l m of ;330 nm) and rather low quantum yield (0.04 to 0.07). The second class consisted of exposed tryptophan residues in a high-polar aqueous environment with long-wavelength po- sition of spectra (l m of ;350 nm) and quantum yield equal or higher than that of free aqueous tryptophan (;0.13– 0.17). This hypothesis had been based on the observation that protein spectrum shifts toward 350 –353 nm upon de- naturation by urea, and toward 330 –332 nm upon addition of anionic detergents in acidic solutions. However, this model could not explain the existence of proteins with a high quantum yield (for example, 0.20 – 0.27 for serum albumins (Longworth, 1971)) and intermediate spectral maximum positions (341.5 nm) for some single-tryptophan- containing proteins, such as human serum albumin (Ivkova et al., 1971). In 1973 one of us, with co-workers, revised and extended Konev’s hypothesis of discrete classes of tryptophan resi- dues in proteins and suggested a new model using some additional spectral parameters and approaches (Burstein et al., 1973). The spectral bandwidth at the half-maximal am- plitude Dl was taken as an additional parameter. The linear relationship was found between values of Dl and maximum position l m for tryptophan and other C-3-substituted indole derivatives in solvents of various polarities. These spectra could be regarded as a series of “elementary” components representing the emission bands of individual tryptophan residues in proteins. Existence of a spectral shift accompa- nying the quenching of protein fluorescence by ionic solutes (NO 3 2 ,I 2 , Cs 1 ) indicated the multicomponent character of a protein emission spectrum. It was demonstrated that the spectra of proteins, which are shifted upon quenching, pos- sess Dl values exceeding those of “elementary” compo- nents. The analysis of the differences between initial spectra of such proteins and those after 20% quenching revealed that the best-quenched components have the longest-wave- length position at ;340 nm in native proteins, which is ;10 nm shorter than that postulated in the two-state model. However, the tryptophans in proteins denatured by urea or guanidinium chloride have a spectrum with l m of ;350 nm. Received for publication 12 January 2001 and in final form 7 June 2001. Address reprint requests to (present address) Dr. Yana K. Reshetnyak, Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Tel.: 817-735-5417; Fax: 817-735- 2133; E-mail: [email protected]. © 2001 by the Biophysical Society 0006-3495/01/09/1710/25 $2.00 1710 Biophysical Journal Volume 81 September 2001 1710 –1734

Transcript of Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The...

Page 1: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

Decomposition of Protein Tryptophan Fluorescence Spectra intoLog-Normal Components. II. The Statistical Proof of Discreteness ofTryptophan Classes in Proteins

Yana K. Reshetnyak and Edward A. BursteinInstitute of Theoretical and Experimental Biophysics, Russia Academy of Sciences, Pushchino, Moscow Region, Russia 142290

ABSTRACT The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores inproteins have been proposed in the model of discrete states (Burstein, E. A., N. S. Vedenkina, and M. N. Ivkova. 1973.Photochem. Photobiol. 18:263–279; Burstein, E. A. 1977a. Intrinsic Protein Luminescence (The Nature and Application). InAdvances in Science and Technology (Itogi Nauki i Tekhniki), Biophysics Vol. 7. VINITI, Moscow [In Russian]; Burstein, E. A.1983. Molecular Biology (Moscow) 17:455–467 [In Russian; English translation]). It was assumed that the existence of the fivemost probable spectral classes of emitting tryptophan residues and differences among the classes were analyzed in termsof various combinations of specific and universal interactions of excited fluorophores with their environment. The develop-ment of stable algorithms of decomposition of tryptophan fluorescence spectra into log-normal components gave us anopportunity to apply two mathematically different algorithms, SImple fitting with Mean-Square criterion (SIMS) and PHase-plot-based REsolving with Quenchers (PHREQ) for the decomposition of a representative set of emission spectra of proteins.Here we present the results of decomposition of tryptophan emission spectra of .100 different proteins, some in variousstructural states (native and denatured, in complexes with ions or organic ligands, in various pH-induced conformations, etc.).Analysis of the histograms of occurrence of .300 spectral log-normal components with various maximum positionsconfirmed the statistical discreteness of several states of emitting tryptophan fluorophores in proteins.

INTRODUCTION

Tryptophan fluorescence is widely used to study the loca-tion, physical and dynamic properties of microenvironmentof indole fluorophores, and the structural features and be-havior of the protein molecule as a whole (Burstein, 1976,1977a, 1983; Lakowicz, 1983; Demchenko, 1986). Depend-ing on the environment of tryptophan residues in proteins,the maximum position (lm) and quantum yield (q) of tryp-tophan fluorescence could vary widely, from 308 to 353 nmand from 0.4 to immeasurably low, respectively.

In 1967 Konev put forward the hypothesis of the exis-tence of two main classes of tryptophan residues in proteins,which possess discrete values of fluorescent parameterslm

andq (Konev, 1967; Volotovski and Konev, 1967). One ofthe classes included tryptophan fluorophores inside the pro-tein in a low-polar hydrophobic environment with a shorter-wavelength position of fluorescent maximum (lm of ;330nm) and rather low quantum yield (0.04 to 0.07). Thesecond class consisted of exposed tryptophan residues in ahigh-polar aqueous environment with long-wavelength po-sition of spectra (lm of ;350 nm) and quantum yield equalor higher than that of free aqueous tryptophan (;0.13–0.17). This hypothesis had been based on the observationthat protein spectrum shifts toward 350–353 nm upon de-

naturation by urea, and toward 330–332 nm upon additionof anionic detergents in acidic solutions. However, thismodel could not explain the existence of proteins with ahigh quantum yield (for example, 0.20–0.27 for serumalbumins (Longworth, 1971)) and intermediate spectralmaximum positions (341.5 nm) for some single-tryptophan-containing proteins, such as human serum albumin (Ivkovaet al., 1971).

In 1973 one of us, with co-workers, revised and extendedKonev’s hypothesis of discrete classes of tryptophan resi-dues in proteins and suggested a new model using someadditional spectral parameters and approaches (Burstein etal., 1973). The spectral bandwidth at the half-maximal am-plitudeDl was taken as an additional parameter. The linearrelationship was found between values ofDl and maximumpositionlm for tryptophan and other C-3-substituted indolederivatives in solvents of various polarities. These spectracould be regarded as a series of “elementary” componentsrepresenting the emission bands of individual tryptophanresidues in proteins. Existence of a spectral shift accompa-nying the quenching of protein fluorescence by ionic solutes(NO3

2, I2, Cs1) indicated the multicomponent character ofa protein emission spectrum. It was demonstrated that thespectra of proteins, which are shifted upon quenching, pos-sessDl values exceeding those of “elementary” compo-nents. The analysis of the differences between initial spectraof such proteins and those after 20% quenching revealedthat the best-quenched components have the longest-wave-length position at;340 nm in native proteins, which is;10nm shorter than that postulated in the two-state model.However, the tryptophans in proteins denatured by urea orguanidinium chloride have a spectrum withlm of ;350 nm.

Received for publication 12 January 2001 and in final form 7 June 2001.

Address reprint requests to (present address) Dr. Yana K. Reshetnyak,Department of Molecular Biology and Immunology, Institute for CancerResearch, University of North Texas Health Science Center, 3500 CampBowie Blvd., Fort Worth, TX 76107. Tel.: 817-735-5417; Fax: 817-735-2133; E-mail: [email protected].

© 2001 by the Biophysical Society

0006-3495/01/09/1710/25 $2.00

1710 Biophysical Journal Volume 81 September 2001 1710–1734

Page 2: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

These results allowed us to develop an extended model ofdiscrete states (classes) of tryptophan residues in proteins,which assumed the existence of five statistically most prob-able classes (Burstein et al., 1973; Burstein, 1977a, 1983).According to the model, the following discrete classes oftryptophan residues were predicted to be most probable inproteins (Burstein, 1977b, 1983):

1. Class A (lm 5 308 nm, structured spectra); the fluoro-phores, which do not form hydrogen-bound complexesin the excited state (exciplexes) (Hershberger et al.,1981) with solvent or neighboring protein groups;

2. Class S (lm 5 316 nm, structured spectra) includes theburied tryptophan residues that can form the exciplexeswith 1:1 stoichiometry;

3. Class I (lm 5 330–332 nm,Dl 5 48–50 nm) repre-sents the buried fluorophores that can form the exci-plexes with 2:1 stoichiometry;

4. Class II (lm 5 340–342 nm,Dl 5 53–55 nm) repre-sents the fluorophores exposed to the bound waterpossessing very long dipole relaxation time, which pre-cludes completing the relaxation-induced spectral shiftduring the excited-state lifetime;

5. Class III (lm 5 350–353 nm,Dl 5 59–61 nm) con-tains rather fully exposed fluorophores surrounded byhighly mobile water completely relaxing during theexcitation lifetime, which makes their spectra almostcoinciding with those of free aqueous tryptophan.

At that time, there was constructed and used the calibrateddiagramDl versuslm for estimating the contributions ofthree most frequent model classes (I, II, and III) of trypto-phan residues in the fluorescence spectrum of a protein(Burstein et al., 1973). The application of this hypothesiswas rather effective in the interpretation of protein trypto-phan fluorescence data in various biophysical and molecu-lar-biology studies. However, the idea of the existing ofdiscrete classes of tryptophans in proteins was based on theanalysis of very limited numbers of proteins. Now it seemsreasonable to reappraise the hypothesis of discrete statesapplying more recent analytic and computing methods tothe wide statistical database of protein spectra.

Such a reappraisal is possible now due to progress inprotein preparation techniques and methods of componentanalysis of protein emission spectra. It was shown that thefluorescence spectra of tryptophan and its derivatives maybe accurately described analytically by a log-normal func-tion (Burstein, 1976), proposed initially for the absorptionspectra by Siano and Metzler (1969). Thereupon, it wasfound that the shape of the emission spectra of tryptophanboth in solutions and in a protein depends only on thespectral maximum positionlm in a known manner (Bursteinand Emelyanenko, 1996). This fact allowed us to developeffective algorithms of reliable decomposition of tryptophanfluorescence spectra of proteins into individual “elementa-

ry” log-normal components (Abornev and Burstein, 1992;Burstein et al., 2001).

Here we present the results of such decomposition oftryptophan emission spectra of.100 different proteins,some in various structural states (native and denatured, incomplexes with ions or organic ligands, in various pH-induced conformations, etc.). The database of obtained log-normal components allowed us to check the hypothesis ofthe discrete classes of tryptophan residues on the basis of arepresentative set of proteins. Analysis of the histograms ofoccurrence of spectral components with various maximumpositions confirmed the statistical discreteness of severalstates of emitting tryptophan fluorophores in proteins. Thediscrete classes observed here are similar to those proposedin the model of discrete states in 1973–1977.

MATERIALS AND METHODS

Fluorescence spectra

The large majority of protein fluorescence spectra used in this work andlisted in Table 1 were taken from the archive of experimental data of theLaboratory of Functional Biophysics of Proteins (Institute of Theoreticaland Experimental Biophysics of the Russia Academy of Sciences). Thespectra have been published and/or analyzed in various special publicationscited in the column “References.” These publications also contain the dataabout protein preparations and techniques of fluorescence measurements. Ifthe spectra were measured elsewhere and given to us, they were recorrectedfor instrument spectral sensitivity curves using the emission spectra ofaqueous tryptophan solution as a standard. Such a recorrection was nec-essary because the component analysis algorithms were developed basedon the model spectra measured with our lab-made instrument (Burstein etal., 1973; Bukolova-Orlova et al., 1974) with a carefully estimated spectralsensitivity curve (Burstein and Emelyanenko, 1996).

The preparations of wheat germ agglutinin, inorganic pyrophosphatase(pyrophosphate phosphohydrolase) from baker’s yeast, monellin fromDi-oscoreophyllum cummensiifruits, Escherichia colialkaline phosphatase,and subtilisin BPN9 produced by Sigma (St. Louis, MO) were kindly givento us by Dr. J. Borejdo (University of North Texas Health Science Center,Fort Worth, TX). Human and bovine carbonic anhydrases II, staphylococ-cal serine protease, and subtilisin Carlsberg (Sigma) were a kind gift fromDr. S. S. Lehrer (Boston Biomedical Research Institute, Boston, MA). Thepreparation of bovine carboxypeptidase A was from Reanal (Hungary).The yeast 3-phosphoglycerate kinase was from Sigma. Porcine pancreaticlipase was from Serva. Hen egg white ovalbumin was from Reakhim(Olaine, Latvia). Actin from dog heart was prepared by one of us (Ya.K. R.) according to Pardee and Spudich (1982).

Fluorescence measurements

Fluorescence spectra of the several proteins marked in Table 1 (in the“References” column) as “Present work” were measured by us during thelast six years. Acrylamide, KCl, KI, CsCl, Tris, and ATP preparations usedin these experiments were ultra-pure grade of Russian or Soviet productionor Sigma.

Steady-state emission spectra were recorded on the lab-made spec-trofluorimeter (Burstein et al., 1973; Bukolova-Orlova et al., 1974) withcollection of emitted light from the cell front face, that allowed applicationof the strict correction function for screening and reabsorption inner filtereffects (Burstein, 1968). The fluorescence was excited with the mercury

Discrete States of Tryptophan Residues 1711

Biophysical Journal 81(3) 1710–1734

Page 3: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

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al.,

1968

Page 4: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Nam

ean

dC

ondi

tions

Pro

tein

Cod

esN

o.of

Trp

sR

efer

ence

s

40C

hym

otry

psin

ogen

A(b

ovin

e);

inw

ater

;pH

7.5

and

pH8.

6C

HG

8B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ivko

vaet

al.,

1968

;R

eshe

tnya

kan

dB

urst

ein,

1997

b41

Ch

ymo

tryp

sin

og

en

A(b

ovi

ne

)in

35

%d

ioxa

ne

CH

G.D

35

8B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ivko

vaet

al.,

1968

42C

hym

otr

ypsi

no

ge

nA

(bo

vin

e)

in6

0–

70

%d

ioxa

ne

CH

G.D

60

-70

8B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ivko

vaet

al.,

1968

43C

hym

otr

ypsi

no

ge

nA

(bo

vin

e);8

Mur

eaC

HG

.UR

8B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ivko

vaet

al.,

1968

44C

reat

ine

phos

phok

inas

e(r

abbi

tsk

elet

alm

uscl

e);

pH6.

0C

RK

23

4B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a

45E

last

ase

(hum

anne

utro

phils

)E

NH

3M

ely

etal

.,19

9746

Fib

rinog

en‡

(bov

ine)

,fr

agm

ent

E;

pH7.

5F

GE

V.

I.E

mel

yane

nko

(thi

sla

b)47

Fib

rone

ctin

;pH

8.0

FB

N39

Kot

elia

nsky

etal

.,19

8148

Fila

min

(hum

an);

pH7.

8F

IL17

Kot

elia

nsky

etal

.,19

8249

Glu

cago

nG

LG1

Aba

bou,

1998

50G

lyce

rald

ehyd

e-3-

phos

phat

ede

hydr

ogen

ase,

apo-

and

holo

-for

ms

(por

cine

skel

etal

mus

cle)

;A

TP

,pH

7.5,

pH8.

5G

PD

3B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ved

enki

naan

dB

urst

ein,

1970

51P

rote

inG

(Str

ep

toco

ccu

ssp.

)G

PS

1A

babo

u,19

9852

He

mo

glo

bin§

(hu

ma

n),

a-c

ha

ins,

red

uce

da

nd

carb

oxy

me

thyl

ate

d;

pH7.

5H

BA

.CM

3A

born

ev,

1993

53Im

mun

oglo

bulin

G(b

ovin

e);

pH6.

6IG

GB

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Sha

nin

etal

.,19

6854

Imm

un

og

lob

ulin

G(b

ovi

ne

);8M

urea

IGG

.UR

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a;S

hani

net

al.,

1968

55In

orga

nic

pyro

phos

phat

ase

(bak

er’s

yeas

t);

pH7.

3IO

P6

Pre

sent

wor

k56

a-L

acta

lbum

in(c

owm

ilk);

1m

MC

aCl

2,

pH7.

4LA

B4

Per

mya

kov

etal

.,19

8557

a-L

acta

lbum

in(h

uman

milk

);1

mM

CaC

l2,

pH7.

4LA

H3

Per

mya

kov

etal

.,19

9158

b-L

acto

glob

ulin

(cow

milk

);pH

2.4,

pH4.

6LG

B.A

C2

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a;K

apla

nas

etal

.,19

73,

1975

59b

-Lac

togl

obul

in(c

owm

ilk);

pH6.

4LG

B.N

2B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Kap

lana

set

al.,

1973

,19

7560

b-L

acto

glob

ulin

(cow

milk

);pH

8.1

LGB

.AL

2B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Kap

lana

set

al.,

1975

61b

-Lac

togl

obul

in¶(B

actr

ian

cam

elm

ilk);

0.01

Mgl

ycin

,pH

2.0–

3.0

LGC

.AC

3A

born

ev,

1993

62b

-Lac

togl

obul

in¶(B

actr

ian

cam

elm

ilk);

pH7.

0LG

C.N

3A

born

ev,

1993

63Li

pase

(por

cine

panc

reas

);pH

7.4

LPP

6P

rese

ntw

ork

64Li

pase

(whe

atge

rm);

pH7.

5LW

GA

born

ev,

1993

65Ly

sozy

me

(hen

egg

whi

te);

HC

l,pH

2.2;

glyc

in,

pH3.

5LZ

C.A

C6

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a66

Lyso

zym

e(h

eneg

gw

hite

);pH

7.4–

7.8

LZC

.N6

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a67

Lys

ozy

me

(he

ne

gg

wh

ite);8

Mur

ea,

pH2.

6,pH

8.0

LZ

C.U

R6

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a68

Mas

topa

ran

(was

pve

nom

)M

SP

1A

babo

u,19

9869

Mel

ittin

,\m

onom

er(A

pis

me

llife

rave

nom

);w

ater

MLT

.MO

N1

Per

mya

kov

and

Dei

kus,

1995

;R

eshe

tnya

kan

dB

urst

ein,

1997

a70

Mel

ittin

,\te

tram

er(A

pis

me

llife

rave

nom

);1.

5–1.

6M

KC

l,pH

7.4

MLT

.TE

TR

431

Per

mya

kov

and

Dei

kus,

1995

;R

eshe

tnya

kan

dB

urst

ein,

1997

a71

Mel

ittin

,\(A

pis

me

llife

rave

nom

)1ca

lmod

ulin

1C

aCl 2,

pH7.

5M

LT.C

AM

1or 43

1P

erm

yako

van

dD

eiku

s,19

95

72M

elitt

in,

mon

omer

(Ap

ism

elli

fera

veno

m)

with

lipos

omes;

2M

NaC

lM

LT.L

S1

Lado

khin

,19

9073

H-m

erom

yosi

n(r

abbi

tsm

ooth

mus

cle)

HM

M11

Vis

hnev

skay

aet

al.,

1976

74L-

mer

omyo

sin,

frac

tion

1,LM

M59

and

LMM

77(r

abbi

tsk

elet

alm

uscl

e)LM

M2

Vis

hnev

skay

aet

al.,

1976

;Z

hou

etal

.,19

9875

Mon

ellin

(Dio

sco

reo

ph

yllu

mcu

mm

en

sii

);pH

7.3

MO

N1

Pre

sent

wor

k76

Myo

glob

in**

(bea

ver

hear

t),

apo-

form

;pH

7.5

MG

B.A

PO

2A

born

ev,

1993

77M

yosi

n(r

abbi

tsk

elet

alm

uscl

e);

0.5

MK

Cl

MS

S20

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a78

Myo

sin

(ra

bb

itsk

ele

talm

usc

le);8

Mur

ea,

pH7.

8M

SS

.UR

20B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a

79M

yosi

nsu

bfra

gmen

t1††

(rab

bit

skel

etal

mus

cle)

;50

mM

KC

l,1

mM

MgC

l 2,

pH7.

5M

S1

5R

eshe

tnya

ket

al.,

2000

80M

yosi

nsu

bfra

gmen

t1††

(ske

leta

lmus

cle,

rabb

it);

50m

MK

Cl,

1m

MM

gCl 2

,0.

2m

MA

TP

,pH

7.5

MS

1.A

TP

5R

eshe

tnya

ket

al.,

2000

Biophysical Journal 81(3) 1710–1734

Page 5: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Nam

ean

dC

ondi

tions

Pro

tein

Cod

esN

o.of

Trp

sR

efer

ence

s

81N

euro

toxi

nI

(cob

raN

aja

na

jao

xia

nav

enom

);pH

4.6–

6.0

NO

12

Buk

olov

a-O

rlova

etal

.,19

74;

Buk

olov

a-O

rlova

etal

.,19

7682

Neu

roto

xin

II(c

obra

Na

jan

aja

oxi

an

aven

om);

pH6.

0N

O2

2B

ukol

ova-

Orlo

vaet

al.,

1974

;B

ukol

ova-

Orlo

vaet

al.,

1976

83N

ucle

ase

(Sta

ph

ylo

cocc

us

au

reu

s);

pH7.

5N

ST

1A

babo

u,19

9884

Nuc

leos

ide

diph

osph

ate

kina

sea‡

‡(r

at,

reco

mbi

nant

);pH

7.7–

9.3

ND

PK

.A.8

3Trp

1T

yrO

2

Orlo

vet

al.,

1997

;O

rlov

etal

.,19

99

85N

ucle

osid

edi

phos

phat

eki

nasea

‡‡

(rat

,re

com

bina

nt);

pH4.

0–5.

5N

DP

K.A

.5.L

IS3

Orlo

vet

al.,

1997

;O

rlov

etal

.,19

9986

Nuc

leos

ide

diph

osph

ate

kina

se a‡

‡(r

at,

reco

mbi

nant

);0.

5–1.

0M

KC

l,pH

4.0–

5.5

ND

PK

.A.5

.HIS

3O

rlov

etal

.,19

97;

Orlo

vet

al.,

1999

87N

ucle

osid

edi

phos

phat

eki

nase b

‡‡

(rat

,re

com

bina

nt);

pH7.

5–9.

5N

DP

K.B

.83

Orlo

vet

al.,

1997

;O

rlov

etal

.,19

9988

Nuc

leos

ide

diph

osph

ate

kina

se b‡

‡(r

at,

reco

mbi

nant

);pH

4.5–

5.2

ND

PK

.B.A

C3

Orlo

vet

al.,

1997

;O

rlov

etal

.,19

9989

Ova

lbum

in(h

eneg

gw

hite

);lo

wio

nic

stre

ngth

,pH

7.4

OV

H.L

IS3

Pre

sent

wor

k90

Ova

lbum

in(h

eneg

gw

hite

);hi

ghio

nic

stre

ngth

,pH

7.4

OV

H.H

IS3

Pre

sent

wor

k91

Pap

ain

(Ca

rica

pa

pa

ya);

glyc

in,

pH3.

1–4.

0P

AP

.AC

5B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ved

enki

naan

dB

urst

ein,

1970

;V

eden

kina

etal

.,19

7192

Pap

ain

(Ca

rica

pa

pa

ya);

wat

er,

pH6.

8–7.

0P

AP

.N5

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a;V

eden

kina

and

Bur

stei

n,19

70;

Ved

enki

naet

al.,

1971

93P

apai

n(Ca

rica

pa

pa

ya);

glyc

in,

pH9.

3–9.

8P

AP

.AL

5B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ved

enki

naan

dB

urst

ein,

1970

;V

eden

kina

etal

.,19

7194

Pa

pa

in(C

arica

pa

pa

ya);8

Mur

ea,

pH6.

2P

AP

.UR

5B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ved

enki

naan

dB

urst

ein,

1970

;V

eden

kina

etal

.,19

7195

Par

valb

umin

II§§

(cod

Ga

du

sm

orr

hu

a),ac

idfo

rm;

pH2.

0P

AC

.AC

1A

born

ev,

1993

96P

arva

lbum

inII§

§(c

odG

ad

us

mo

rrh

ua),

apo-

form

;pH

7.7

PA

C.A

PO

1A

born

ev,

1993

97P

arva

lbum

inII§

§(c

odG

ad

us

mo

rrh

ua),

Mg-

form

;1

mM

MgC

l 2,pH

7.7

PA

C.M

G1

Abo

rnev

,19

9398

Par

valb

umin

II§§

(cod

Ga

du

sm

orr

hu

a),C

a-fo

rm;

1m

MC

aCl 2,

pH7.

7P

AC

.CA

1A

born

ev,

1993

99P

arva

lbum

in(w

hitin

gGa

du

sm

erla

ng

us),

apo-

form

;pH

6.4

PA

M.A

PO

1P

erm

yako

vet

al.,

1980

100

Par

valb

umin

(whi

tingG

ad

us

me

rla

ng

us);

m0.

5M

,pH

8.8

PA

M1

Per

mya

kov

etal

.,19

8010

1P

arva

lbum

in§§

(Ala

ska

polla

ck,T

he

rag

rach

alc

og

ram

ma),

apo-

form

;pH

8.0

PM

T.A

PO

1V

.I.

Em

elya

nenk

o(t

his

lab)

102

Par

valb

umin§

§(A

lask

apo

llack

,Th

era

gra

cha

lco

gra

mm

a),C

a-fo

rm;

1m

MC

aCl 2,

pH8.

0P

MT

.CA

1V

.I.

Em

elya

nenk

o(t

his

lab)

103

Par

valb

umin§

§(A

lask

apo

llack

,Th

era

gra

cha

lco

gra

mm

a),M

g-fo

rm;

1m

MM

gCl 2,

pH8.

0P

MT

.MG

1V

.I.

Em

elya

nenk

o(t

his

lab)

104

Par

valb

umin§

§(P

acifi

cna

vaga

,Ele

gin

us

gra

cilis),

apo-

form

;pH

8.0

PA

N.A

PO

1V

.I.

Em

elya

nenk

o(t

his

lab)

105

Par

valb

umin§

§(P

acifi

cna

vaga

,Ele

gin

us

gra

cilis),

Ca-

form

;1

mM

CaC

l 2,pH

8.0

PA

N.C

A1

V.

I.E

mel

yane

nko

(thi

sla

b)

106

Par

valb

umin§

§(P

acifi

cna

vaga

,Ele

gin

us

gra

cilis),

Mg-

form

,1

mM

MgC

l 2,pH

8.0

PA

N.M

G1

V.

I.E

mel

yane

nko

(thi

sla

b)

107

Pep

sin

(bov

ine)

;pH

1.3–

2.8

PE

B.1

-35

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a10

8P

epsi

n(b

ovin

e);

wat

eror

pH4.

0–5.

2P

EB

.4-5

5B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a

109

Pe

psi

n(b

ovi

ne

);pH

5.2–

9.0

PE

B.6

-95

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a11

0P

ep

sin

(bo

vin

e);8

Mur

ea,

pH5.

2P

EB

.UR

5B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a

111

Pho

spha

tase

alka

line

(E

.co

li);

pH7.

1P

HA

233

Pre

sent

wor

k11

23-

Pho

spho

glyc

erat

eki

nase

(yea

st);

pH7.

4P

GK

2P

rese

ntw

ork

113

Pho

spho

lipas

eA 2(b

ovin

epa

ncre

as);

pH6.

0,pH

7.4

PLB

1P

erm

yako

van

dD

eiku

s,19

9511

4P

hosp

holip

ase

A 2(p

orci

nepa

ncre

as);

pH7.

4P

LS1

Per

mya

kov

and

Dei

kus,

1995

Biophysical Journal 81(3) 1710–1734

Page 6: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Nam

ean

dC

ondi

tions

Pro

tein

Cod

esN

o.of

Trp

sR

efer

ence

s

115

Pho

spho

lipas

eA 2(c

obra

Na

jan

aja

oxi

an

aven

om);

low

ioni

cst

reng

th,

pH7.

5P

AO

.LIS

3O

rlova

,19

79

116

Pho

spho

lipas

eA 2(c

obra

Na

jan

aja

oxi

an

aven

om);

high

ioni

cst

reng

th,

pH7.

5P

AO

.HIS

3O

rlova

,19

79

117

Ph

osp

ho

lipa

seA 2

(co

bra

Na

jan

aja

oxi

an

ave

no

m);6

MG

dnH

Cl,

pH7.

5P

AO

.GD

N3

Orlo

va,

1979

118

Pho

spho

lipas

eC

,ph

osph

atid

ylch

olin

e-sp

ecifi

c,ho

lo-f

orm

(B

ac.

cere

us);

pH7.

1P

LC9

D.

B.

Vep

rints

ev(t

his

lab)

119

Pro

lact

in(b

ovin

e);

pH8.

5P

RL

2P

erm

yako

vet

al.,

1997

120

Pro

pion

ylch

olin

ees

tera

se(h

uman

);pH

7.5

PC

EA

born

ev,

1993

121

HIV

-1pr

otea

se;

low

ioni

cst

reng

th,

T5

202

67°C

,0–

50%

glyc

erol

,pH

8.0

PR

H.L

IS2

12

A.

I.K

orne

lyuk

¶¶

122

HIV

-1pr

otea

se;

high

ioni

cst

reng

th,

pH8.

0P

RH

.HIS

21

2A

.I.

Kor

nely

uk¶¶

123

HIV

-1pr

otea

sew

ithpe

psta

tin,

pH8.

0P

RH

.PS

T2

12

A.

I.K

orne

lyuk

¶¶

124

Ser

ine

prot

ease

(S

tap

hyl

oco

ccu

sa

ure

us

);pH

7.5

PR

S2

Pre

sent

wor

k12

5P

rote

ase

K(Tritir

ach

ium

alb

um

Lim

ber)

;pH

7.5

PR

K2

Res

hetn

yak

and

Bur

stei

n,19

97a

126

Pyr

uvat

eki

nase

(rab

bit

skel

etal

mus

cle)

;pH

7.5

PY

K3

Abo

rnev

,19

9312

7P

yruv

ate

kina

se(r

abbi

tsk

elet

alm

uscl

e)P

EG

conj

ugat

eP

YK

.PE

G3

Abo

rnev

,19

9312

8R

ecov

erin

MP

26,

apo-

form

;pH

8.1

RE

C.A

PO

3S

.E

.P

erm

yako

v(t

his

lab)

129

Rec

over

inM

P26

1C

aCl 2,

pH8.

1R

EC

.CA

3S

.E

.P

erm

yako

v(t

his

lab)

130

Red

cells

ghos

ts(h

uman

);pH

7.8

RC

GO

rlova

,19

7913

1R

ibon

ucle

ase

C 2(A

spe

rgill

us

cla

vatu

s);0–

20%

glyc

erol

,pH

4.0,

pH6.

0,pH

8.0–

8.4

RN

C1

Bur

stei

n,19

77a;

Bur

stei

n,19

83;

Gris

hche

nko

etal

.,19

76

132

Rib

onuc

leas

ein

trac

ellu

lar

\\(A

spe

rgill

us

cla

vatu

s);pH

8.0

RN

IV

.M

.G

rishc

henk

o(t

his

lab)

133

RN

Ase

T 1**

*(

Asp

erg

illu

so

ryza

e);20

%gl

ycer

ol,

pH7–

8R

NT

1B

urst

ein,

1977

a;B

urst

ein,

1983

;G

rishc

henk

oet

al.,

1976

134

Rib

osom

alpr

otei

nS

4†

††

(E.

coli)

;pH

7.0

RS

41

T.

L.B

ushu

eva

(thi

sla

b)13

5R

ibos

omal

prot

ein

S7

††

†(E

.co

li);

pH7.

0R

S7

2B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a

136

Rib

osom

e-in

activ

atin

gpr

otei

n‡

‡‡

(ric

inag

glut

inin

,Ric

inu

sco

mm

un

is);pH

4.0–

7.0

RIP

T.

L.B

ushu

eva§§

§

137

Ric

inch

ain

A(R

icin

us

com

mu

nis);

pH4.

0–7.

0R

CA

1B

ushu

eva

and

Ton

evits

ky,

1987

a,b;

Bus

huev

aan

dT

onev

itsky

,19

88;

Bus

huev

aet

al.,

1990

138

Ric

inch

ain

B(R

icin

us

com

mu

nis);

pH4.

0R

CB

.49

Bus

huev

aan

dT

onev

itsky

,19

87a,

b;B

ushu

eva

and

Ton

evits

ky,

1988

;B

ushu

eva

etal

.,19

9013

9R

icin

chai

nB

(Ric

inu

sco

mm

un

is);pH

5.0–

7.0

RC

B.5

-79

Bus

huev

aan

dT

onev

itsky

,19

87a,

b;B

ushu

eva

and

Ton

evits

ky,

1988

;B

ushu

eva

etal

.,19

9014

0R

icin

chai

nB

(Ric

inu

sco

mm

un

is)1

lact

ose;

pH4.

0R

CB

.4.L

AC

9B

ushu

eva

and

Ton

evits

ky,

1987

a,b;

Bus

huev

aan

dT

onev

itsky

,19

88;

Bus

huev

aet

al.,

1990

141

Ric

in(R

icin

us

com

mu

nis)

(cha

inA

1ch

ain

B)

with

and

with

out

lact

ose;

pH4.

0–7.

0R

CC

119

Bus

huev

aan

dT

onev

itsky

,19

87a,

b;B

ushu

eva

and

Ton

evits

ky,

1988

;B

ushu

eva

etal

.,19

9014

2S

omat

otro

pin,

grow

thho

rmon

e(h

uman

);pH

8.2

ST

P1

Per

mya

kov

and

Dei

kus,

1995

143

Spe

ctrin

,a-c

hain

andb

-cha

inS

PC

42142

T.

L.B

ushu

eva§§

§

144

Sub

tilis

inB

PN9

;pH

7.3

BP

N3

Pre

sent

wor

k14

5S

ubtil

isin

Car

lsbe

rg;

pH7.

5S

LC1

Pre

sent

wor

k14

6T

elok

inK

RP

,m

yosi

nlig

ht-c

hain

kina

se(c

hick

engi

zzar

d)K

RP

1B

ushu

eva

etal

.,19

9914

7T

ob

acc

om

osa

icvi

rus,

pro

tein

A;8M

urea

TM

V.U

R3

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a;V

eden

kina

and

Bur

stei

n,19

7014

8T

ropo

nin

T(r

abbi

tsk

elet

alm

uscl

e);

pH6.

8T

NT

2M

oroz

ova

etal

.,19

8814

9T

ryps

in(b

ovin

e);

wat

er,

pH7.

3,pH

8.7

TR

Y4

Bur

stei

net

al.,

1973

;B

urst

ein,

1977

a;V

eden

kina

and

Bur

stei

n,19

70;

Ivko

vaet

al.,

1968

;R

eshe

tnya

kan

dB

urst

ein,

1997

b

Biophysical Journal 81(3) 1710–1734

Page 7: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Nam

ean

dC

ondi

tions

Pro

tein

Cod

esN

o.of

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sR

efer

ence

s

150

Try

psi

n(b

ovi

ne

)in

35

%d

ioxa

ne

TR

Y.D

35

4B

urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

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enki

naan

dB

urst

ein,

1970

;Iv

kova

etal

.,19

6815

1T

ryp

sin

(bo

vin

e)

in6

0–

70

%d

ioxa

ne

TR

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60

-70

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urst

ein

etal

.,19

73;

Bur

stei

n,19

77a;

Ved

enki

naan

dB

urst

ein,

1970

;Iv

kova

etal

.,19

6815

2T

ryps

inog

en(b

ovin

e);

pH7.

4T

RG

4R

eshe

tnya

kan

dB

urst

ein,

1997

b15

3V

incu

lin¶¶¶(h

uman

)V

IN10

Bus

huev

a§§

§

154

Vin

culin

¶¶¶

(hu

ma

n);

8M

urea

VIN

.UR

10B

ushu

eva§§

§

155

Vip

oxin

,pr

otei

nA

(Vip

era

am

mo

dyt

es

am

mo

dyt

es

);pH

3.8–

4.8

VT

A.A

C1

Buk

olov

a-O

rlova

etal

.,19

79;

Buk

olov

a-O

rlova

etal

.,19

80;

Tch

orba

nov

etal

.,19

7715

6V

ipox

in,

prot

ein

A(V

ipe

raa

mm

od

yte

sa

mm

od

yte

s);

high

ioni

cst

reng

th,

pH7.

2–8.

0V

TA

.HIS

231

Buk

olov

a-O

rlova

etal

.,19

79;

Buk

olov

a-O

rlova

etal

.,19

80;

Tch

orba

nov

etal

.,19

7715

7V

ipox

in,

prot

ein

A(V

ipe

raa

mm

od

yte

sa

mm

od

yte

s);

low

ioni

cst

reng

th,

pH7.

2–8.

0V

TA

.LIS

1B

ukol

ova-

Orlo

vaet

al.,

1979

;B

ukol

ova-

Orlo

vaet

al.,

1980

;T

chor

bano

vet

al.,

1977

158

Vip

oxin

,pr

otei

nB

(Vip

era

am

mo

dyt

es

am

mo

dyt

es

);pH

3.6,

pH6.

2–8.

1V

TB

2B

ukol

ova-

Orlo

vaet

al.,

1979

;B

ukol

ova-

Orlo

vaet

al.,

1980

;T

chor

bano

vet

al.,

1977

159

Vip

oxin

,co

mpl

exA

1B

(Vip

era

am

mo

dyt

es

am

mo

dyt

es

);pH

4.8,

pH8.

0V

TC

112

Buk

olov

a-O

rlova

etal

.,19

79;

Buk

olov

a-O

rlova

etal

.,19

80;

Tch

orba

nov

etal

.,19

7716

0V

iscu

min

(le

ctin

ML

-1;

mis

leto

eV

iscu

ma

lbu

m);pH

4.0

VS

C.A

C21

9B

ushu

eva

and

Ton

evits

ky,

1988

;B

ushu

eva

etal

.,19

8816

1V

iscu

min

(lect

inM

L-1;

mis

leto

eVis

cum

alb

um)

;pH

5.0–

7.5

VS

C.5

-721

9B

ushu

eva

and

Ton

evits

ky,

1988

;B

ushu

eva

etal

.,19

8816

2V

iscu

min

(le

ctin

ML

-1;

mis

leto

eV

iscu

ma

lbu

m),

mo

difi

ed

by

eth

yla

mm

on

ium

nitr

ate

;pH4.

0,pH

7.0

VS

C.M

OD

219

Bus

huev

aan

dT

onev

itsky

,19

88;

Bus

huev

aet

al.,

1988

163

Vis

cum

in(le

ctin

ML

-1;

mis

leto

eV

iscu

ma

lbu

m),

mo

difi

ed

by

eth

yla

mm

on

ium

nitr

ate

;8M

urea

,pH

4.0,

pH7.

0V

SC

.UR

219

Bus

huev

aan

dT

onev

itsky

,19

88;

Bus

huev

aet

al.,

1988

Den

atur

edpr

otei

nco

des

appe

arin

italic

s.*P

rese

nted

byD

r.A

ndrz

ejS

obot

a,N

.N

enck

iIns

titut

eof

Exp

erim

enta

lBio

logy

,W

arsa

w,

Pol

and

(Ban

doro

wic

zet

al.,

1992

;S

obot

aet

al.,

1993

).†P

repa

red

and

pres

ente

dby

Dr.

G.

Ben

zona

na(B

enzo

nana

etal

.,19

74;

Cox

etal

.,19

76).

‡P

rese

nted

byD

r.L.

V.

Med

ved,

Inst

itute

ofB

ioch

emis

try,

Ukr

aine

Aca

dem

yof

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ence

,K

iev.

§P

repa

red

and

pres

ente

dby

Dr.

L.V

.A

batu

rov,

Inst

itute

ofM

olec

ular

Bio

logy

,R

AS

,M

osco

w.

¶ Pre

pare

dby

Dr.

L.P

.K

alin

iche

nko

(thi

sla

b)fr

omca

mel

milk

pres

ente

dby

Dr.

D.

Nya

maa

,In

stitu

teof

Bio

tech

nolo

gy,

Mon

golia

nA

cade

my

ofS

cien

ce,

Ul

anB

aata

r,M

ongo

lia.

\ Pre

sent

edby

Dr.

A.

P.

Dem

chen

ko,

Inst

itute

ofB

ioch

emis

try,

Nat

iona

lAca

dem

yof

Sci

ence

ofU

krai

ne,

Kie

v.**

Pre

sent

edby

Dr.

R.

I.A

rtyu

khan

dD

r.E

.A

.S

hlya

pnik

ova,

Inst

itute

ofT

heor

etic

alan

dE

xper

imen

talB

ioph

ysic

s,R

AS

,P

ushc

hino

.†

† Pre

pare

dby

Dr.

O.

And

reev

,U

nive

rsity

ofN

orth

Tex

as,

For

tW

orth

,T

exas

.‡

‡ Pre

pare

dan

dpr

esen

ted

byD

r.N

.Y

a.O

rlov,

this

lab.

§§ P

repa

red

byD

r.L.

P.

Kal

inic

henk

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his

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,us

ing

mod

ified

Hai

ech

met

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ech

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.,19

79).

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rese

nted

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.I.

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nely

uk,

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itute

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s,N

atio

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itute

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icro

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R,

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hchi

no.

***P

rese

nted

byD

r.C

.A

.G

hiro

n.†

†† P

repa

red

and

pres

ente

dby

Dr.

A.

T.

Gud

kov,

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itute

ofP

rote

inR

esea

rch,

Aca

dem

yof

Sci

ence

US

SR

,P

ushc

hino

.‡

‡‡ P

repa

red

byD

r.A

.G

.T

onev

itsky

(Ton

evits

kyet

al.,

1996

).§

§§ S

pect

ram

easu

red

byD

r.T

.L.

Bus

huev

a,C

ardi

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yR

esea

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ter,

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sian

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dem

yof

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ical

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ence

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osco

w.

¶¶¶ P

repa

red

byD

r.V

.E

.K

otel

yans

ky(W

elle

ret

al.,

1990

);sp

ectr

aw

ere

mea

sure

dby

Dr.

T.

L.B

ushu

eva,

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diol

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earc

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ente

r,R

ussi

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cow

.

Biophysical Journal 81(3) 1710–1734

Page 8: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

line 296.7 nm from an SVD-120A mercury lamp isolated with a mono-chromator. The use of the mercury lamp with narrow spectral lines inemission provided the possibility of rather simple correction for the straylight presented in the registered emission spectra. The spectral width ofslits for excitation and emission did not exceed 2 nm. After correction forthe instrument spectral sensitivity (Burstein and Emelyanenko, 1996), theintensities were proportional to the number of photons emitted per unitwavelength interval during a unit time interval. Spectra of almost everyprotein were measured in the presence of varying concentrations of exter-nal quenchers (acrylamide, KI, CsCl, and/or NaNO3). When using ionicquenchers (I2, Cs1, or NO3

2) the total ionic strength was kept constant(from 0.4 to 0.6 M in various experiments) by addition of KCl. Inexperiments with acrylamide the intensities in fluorescence spectra werecorrected for the screening inner filter effect of this quencher at theexcitation wavelength (Burstein, 1968).

Analysis and representation of spectral data

The decomposition of tryptophan fluorescence spectra of proteins intolog-normal components was performed for sets of spectra measured atvarying concentrations of quenchers using two algorithms: SIMS (SImplefitting with Mean-Square criterion) (Abornev and Burstein, 1992; Bursteinet al., 2001) and PHREQ (PHase-plot-basedREsolving with Quenchers,for two-component decomposition) (Burstein et al., 2001). For the decom-position of a single spectrum (without quenchers) the modified SIMSalgorithm was applied (Burstein et al., 2001). Using SIMS, the spectrawere independently fitted by one, two, or three components. The criterionof attaining the solution (a minimal sufficient number of componentsdescribing the spectra) was the minimal root-mean-square differences(residuals) between theoretical and experimental spectra multiplied by thenumber of parameters under fitting. For several proteins two solutions withdifferent numbers of components, which had the similar values of thiscriterion, were selected as the best ones.

The fact that the components of composite tryptophan emission spectraof proteins are wide (;30–60 nm at half-maximal level) compared withthe differences between the values of their maximum positions (from 5 to;50 nm), might considerably reduce the reliability of decomposition.Therefore, we present in Table 2 the averaged results (values of themaximum position,lm(i) and the contribution into the area under spectrum,S(i) and the standard deviations of averaging) obtained separately with themathematically different algorithms: PHREQ and SIMS. The good agree-ment between the parameters that were obtained with these two methodsdemonstrate the reliability of a result. We can note that for the absolutemajority of proteins the results obtained with these two methods differ lessthan by errors of means. It allowed us to present unified results of thecomponent analysis for individual proteins independent of the decompo-sition method.

Moreover, the majority of protein spectra were measured in the pres-ence of various quenchers, in most cases more than once, using prepara-tions from different sources or varying solvent conditions. To extractunified values of the parameters for any given protein or its conformer, twoaveraging schemes were applied to the sets of decomposition results:“Averaging” and “Mean Best Fit” (Table 2). According to the first scheme,the parameters of components were calculated as averages of all solutionsobtained for all individual experiments, independent of the quality of thedecomposition. Within the second scheme, only the data obtained withminimal values of the decomposition functional for each experiment wereaveraged.

RESULTS

In Table 2 we present the results of decomposition oftryptophan fluorescence spectra of.100 different proteins,some in various structural states (native and denatured, in

complexes with ions or organic ligands, and in variouspH-induced states), obtained by the above-mentioned algo-rithms, SIMS and PHREQ, and different averaging schemesof the obtained solutions, averaging and mean best fit. TheSIMS algorithm was used to the log-normal decompositionof 163 sets of tryptophan emission spectra. The PHREQalgorithm was applied to the 105 sets of fluorescence spec-tra because the other 58 spectra of proteins were measuredwithout quenchers, and only the modified SIMS algorithmcould be applied to decompose them. The results obtainedwith both SIMS and PHREQ methods for absolute majorityof proteins differ less than errors of means presented in thelm(i) and S(i) columns. It allows presenting the unifiedresults of the component analysis independent of the de-composition method. Besides the results of log-normalcomponent analysis, Table 2 contains the maximum posi-tion values of whole, non-decomposed fluorescence spectraof proteins (“lm total,” the third column). The denaturedproteins are italicized in the tables.

The database of log-normal components of protein tryp-tophan fluorescence spectra shown in Table 2 may be re-garded as a generalized representative database of fluores-cence spectra of individual tryptophan residues in proteins.It allowed us to perform statistical testing of the hypothesisof discrete states, i.e., the existence of the set of severalclasses of tryptophan residues in proteins discretely differ-ing in their fluorescence spectral maximum positions, whichwas put forward as early as 1973–1977 (Burstein et al.,1973; Burstein, 1977a, b, 1983). Such a test is performedhere by constructing and analyzing the histograms of oc-currence of log-normal components presented in the data-base of Table 2.

Before examining histograms of occurrence of individuallog-normal components, it seems reasonable to considerhow randomly are distributed spectral maximum positionsof whole, non-decomposed emission spectra of proteins ofour database. The histograms of occurrence oflm values ofwhole spectra are shown in Fig. 1,A andB for native anddenatured proteins, respectively, constructed using the “lm

total” values from Table 2. It is seen that the histogram oflm for native proteins is rather well fitted by normal distri-bution with the peak at 336.76 0.4 nm ands 5 6.0 6 0.9nm, which is a good confirmation of random character ofthe excerpt of native proteins. A worse fitting by the normaldistribution was obtained for the maximum positions offluorescence spectra of denatured proteins (the peak at346.56 0.6 nm;s 5 4.16 1.3 nm); however, it is expecteddue to the small number of proteins and, mainly, due toessentially different structural features of protein statesformed under various denaturing effectors used (urea, diox-ane, extreme pH, etc.).

The canonical histograms of occurrence of maximumposition values of log-normal components (with the 2-nmsteps) of emission spectra of native proteins are presented inFig. 2. PanelsA and B represent the distributionslm(i)

1717Biophysical Journal Volume 81 September 2001 1710–1734

Page 9: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

TA

BLE

2T

hed

atab

ase

of

par

amet

ers

of

log

-no

rmal

com

po

nent

so

bta

ined

asre

sult

of

dec

om

po

siti

on

of

tryp

top

han

fluo

resc

ence

spec

tra

of

pro

tein

sus

ing

the

SIM

San

dP

HR

EQ

alg

ori

thm

s

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

1A

CH

335.

460.

51

324.

860.

239

.26

3.5

132

1.66

6.0

46.7

618

.31

322.

664.

936

.66

4.0

132

2.66

3.7

36.8

66.

92

344.

860.

260

.86

3.5

234

2.56

0.7

53.3

618

.32

342.

961.

063

.46

4.0

234

3.06

1.6

63.2

66.

92

AC

D.F

329.

760.

61

317.

962.

548

.46

0.1

132

6.06

5.0

78.4

624

.41

325.

065.

479

.86

21.7

132

7.86

1.6

88.4

67.

02

335.

862.

751

.66

0.1

234

2.76

5.8

21.6

624

.42

341.

965.

920

.26

21.7

234

2.56

2.0

11.6

67.

03

AC

D.G

329.

960.

91

328.

361.

210

060

132

9.96

1.0

1006

01

329.

461.

310

060

132

9.26

1.0

1006

04

AC

R.F

330.

560.

51

329.

864.

510

060

133

0.26

0.5

1006

01

330.

360.

410

060

133

0.56

0.5

1006

05

AC

R.F

.UR

35

0.06

0.5

13

48

.56

3.2

10

060

13

49

.96

0.4

10

060

13

49

.66

1.0

10

060

13

50

.06

0.5

10

060

6A

CR

.G32

9.66

0.5

132

1.06

5.3

47.9

61.

91

320.

960.

535

.16

2.0

132

1.06

0.1

41.5

69.

11

320.

960.

535

.16

2.0

233

5.56

4.5

52.1

61.

62

332.

860.

564

.96

4.0

233

4.06

1.0

58.5

69.

12

332.

860.

564

.96

4.0

7A

CM

334.

761.

21

325.

666.

451

.26

14.9

132

5.26

5.6

53.8

610

.71

325.

465.

650

.76

11.0

132

6.06

6.2

52.2

68.

22

343.

261.

748

.86

14.9

234

3.06

2.5

46.2

610

.72

343.

462.

149

.36

11.0

234

4.46

2.6

47.8

68.

28

AC

M.U

R3

47

.860

.91

33

3.4

61

0.3

15

.76

2.1

13

39

.66

9.6

37

.26

9.9

13

36

.56

8.9

26

.56

13

.71

33

7.7

61

0.5

25

.26

16

.52

35

1.3

60

.28

4.4

62

.12

35

3.7

61

.46

2.8

69

.92

35

2.5

61

.67

3.5

61

3.7

23

51

.76

0.9

74

.86

16

.59

AC

Y33

5.56

0.5

133

0.06

2.9

60.6

60.

11

332.

960.

684

.26

5.2

133

1.36

1.8

70.6

614

.11

331.

862.

970

.66

14.1

234

5.66

3.1

39.4

60.

22

353.

362.

915

.86

5.2

234

8.46

4.0

29.4

614

.12

348.

463.

129

.46

14.1

10A

WG

(1-c

omp.

)34

9.06

0.3

134

9.16

0.2

1006

01

349.

160.

310

060

134

9.16

0.2

1006

01

349.

160.

210

060

(2-c

omp.

)1

333.

462.

25.

560.

81

330.

666.

48.

667.

41

331.

465.

57.

366.

31

330.

467.

14.

361.

42

350.

060.

394

.56

0.8

235

0.66

1.0

91.4

67.

42

350.

460.

892

.46

6.3

234

9.96

0.1

95.7

61.

411

A1A

T33

4.86

0.5

——

132

4.46

0.1

38.9

60.

21

324.

460.

138

.96

0.2

132

4.46

0.1

38.9

60.

22

340.

160.

161

.16

0.2

234

0.16

0.1

61.1

60.

22

340.

160.

161

.16

0.2

12A

SB

.F33

3.06

0.5

131

4.76

2.8

15.3

60.

41

319.

960.

520

.76

1.0

131

7.36

3.7

18.0

63.

81

314.

762.

815

.36

0.4

233

5.16

3.0

84.7

60.

12

335.

660.

579

.36

3.0

233

5.46

0.4

82.0

63.

82

335.

163.

084

.76

0.1

13A

SB

.N34

2.86

0.2

134

2.36

0.5

1006

01

342.

960.

210

060

134

2.76

0.4

1006

01

342.

860.

210

060

14A

SB

.N.IB

338.

660.

51

338.

563.

610

060

133

6.66

1.8

1006

01

337.

661.

710

060

133

8.56

3.6

1006

015

AS

H.F

334.

860.

51

323.

863.

742

.66

0.6

132

8.66

0.5

31.3

62.

01

326.

263.

455

.76

18.5

132

3.86

3.7

42.6

60.

62

342.

563.

657

.46

0.5

234

8.56

0.5

68.7

62.

02

345.

564.

244

.36

18.5

234

2.56

3.6

57.4

60.

516

AS

H.N

341.

360.

51

328.

762.

920

.06

0.1

132

5.76

5.4

19.4

615

.31

326.

764.

225

.16

7.2

132

8.76

2.9

20.0

60.

12

344.

863.

180

.06

0.1

234

5.16

1.8

80.6

615

.32

345.

061.

374

.96

7.2

234

4.86

3.1

80.0

60.

117

AS

H.N

.IB33

8.56

0.5

132

6.26

3.4

40.6

60.

41

327.

160.

546

.16

1.0

132

6.76

0.6

43.4

63.

91

326.

263.

440

.66

0.4

234

6.86

3.2

59.4

60.

32

347.

360.

553

.96

2.0

234

7.16

0.4

56.6

63.

92

346.

863.

259

.46

0.3

18

AS

H.U

R3

45

.663

.81

32

3.1

64

.92

1.5

66

.91

32

5.4

60

.53

5.2

61

.01

32

3.8

63

.72

6.0

69

.31

32

6.0

60

.82

5.9

61

3.2

23

50

.56

4.9

78

.56

6.9

23

48

.46

0.5

64

.86

1.0

23

49

.86

3.7

74

.06

9.3

23

52

.16

4.0

74

.16

13

.219

AD

Y34

4.16

0.5

133

1.16

2.9

55.7

60.

51

330.

861.

635

.76

6.4

133

0.96

1.1

42.4

612

.41

331.

162.

955

.76

0.5

235

6.26

3.2

44.3

60.

52

356.

661.

264

.36

6.4

235

6.46

0.9

57.6

612

.42

356.

263.

244

.36

0.5

20A

TR

N.T

RP

.33

8.16

0.1

133

0.06

1.0

47.5

67.

91

333.

063.

857

.06

30.2

133

2.36

3.0

55.7

629

.71

332.

363.

055

.76

29.7

A2-

K2

(2-

com

p.)

234

4.76

1.5

52.5

67.

92

345.

763.

843

.06

30.2

234

4.46

3.3

44.3

629

.72

344.

463.

344

.36

29.7

(3-c

omp.

)—

—1

318.

460.

55.

063.

01

317.

761.

16.

061.

41

318.

460.

55.

063.

02

336.

360.

552

.06

6.0

233

7.06

0.9

66.1

619

.92

336.

360.

552

.06

6.0

334

1.96

0.5

43.0

610

.03

345.

765.

327

.96

21.4

334

1.96

0.5

43.0

610

.021

AT

RN

.TR

P.

338.

460.

51

330.

363.

341

.06

0.7

133

1.76

4.0

47.4

633

.81

331.

262.

945

.16

24.4

133

0.36

3.3

41.0

60.

7E

11E

22

343.

963.

259

.06

0.7

234

4.76

4.9

52.6

633

.82

344.

463.

554

.96

24.4

234

3.96

3.2

59.0

60.

72

2A

TR

N.T

RP

.UR

35

0.36

0.5

13

16

.56

3.1

0.4

69

.41

34

0.0

60

.51

4.7

67

.01

33

2.4

61

3.8

8.9

61

2.0

13

16

.56

3.1

0.4

69

.42

35

0.4

63

.19

9.6

60

.12

35

2.6

61

.58

5.3

67

.02

35

1.9

61

.39

1.1

61

2.0

23

50

.46

3.1

99.6

60.

1

Page 10: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

23A

TR

N.T

YR

.40

338.

161.

01

326.

563.

235

.86

3.5

132

8.86

3.5

41.6

64.

31

328.

063.

538

.76

4.8

132

6.76

3.0

37.3

65.

22

345.

061.

464

.26

3.5

234

6.46

2.5

58.4

64.

32

345.

962.

261

.36

4.8

234

5.16

1.3

62.7

65.

224

AT

RN

.TY

R.5

933

8.46

0.3

132

7.76

2.6

27.0

68.

51

328.

364.

731

.76

19.1

132

8.06

3.5

29.3

613

.91

324.

767.

629

.66

17.1

234

1.96

0.6

73.0

68.

52

344.

362.

468

.36

19.1

234

2.76

1.9

70.7

613

.92

342.

463.

470

.46

17.1

25A

MY

333.

46

1.4

132

7.96

1.2

63.0

613

.61

330.

561.

779

.16

5.4

132

9.66

1.9

72.5

613

.61

327.

961.

263

.06

13.6

235

0.46

6.9

37.0

613

.62

354.

364.

320

.96

5.4

235

2.86

5.3

27.5

613

.62

350.

466.

937

.06

13.6

26A

X6

(2-c

omp.

)33

1.36

0.5

132

5.56

2.9

67.7

60.

11

326.

060.

574

.06

1.0

132

5.86

0.4

70.9

64.

51

325.

562.

967

.76

0.1

234

6.56

3.1

32.3

60.

12

347.

760.

526

.06

2.0

234

7.16

0.8

29.1

64.

52

346.

563.

132

.36

0.1

(3-c

omp.

)—

—1

317.

260.

510

.36

3.0

131

7.26

0.5

10.3

63.

01

317.

260.

510

.36

3.0

232

9.46

0.5

75.6

63.

02

329.

460.

575

.66

3.0

232

9.46

0.5

75.6

63.

03

356.

360.

514

.26

2.0

335

6.36

0.5

14.2

62.

03

356.

360.

514

.26

2.0

27A

SP

.AC

329.

461.

0—

—1

327.

261.

491

.76

7.8

132

7.26

1.4

91.7

67.

81

327.

261.

491

.76

7.8

235

5.46

3.9

8.36

7.8

235

5.46

3.9

8.36

7.8

235

5.46

3.9

8.36

7.8

28A

SP

.N32

4.76

1.6

132

5.26

1.2

1006

01

324.

561.

810

060

132

4.56

1.7

1006

01

323.

961.

810

060

29A

ZU

.77K

307.

963.

6—

—1

307.

964.

410

060

130

7.96

4.4

1006

01

307.

964.

410

060

30C

BC

338.

360.

21

327.

863.

939

.06

11.4

132

3.36

1.6

27.3

63.

91

325.

663.

633

.16

10.0

132

3.36

1.6

27.3

63.

9(2

-com

p.)

234

4.46

1.7

61.0

611

.42

342.

360.

672

.76

3.9

234

3.36

1.7

66.7

610

.02

342.

360.

672

.76

3.9

(3-c

omp.

)—

—1

315.

565.

514

.96

4.7

131

5.56

5.5

14.9

64.

71

315.

565.

514

.96

4.7

233

7.26

1.9

61.0

63.

22

337.

261.

961

.06

3.2

233

7.26

1.9

61.0

63.

23

347.

861.

124

.16

7.1

334

7.86

1.1

24.1

67.

13

347.

861.

124

.16

7.1

31C

DS

349.

360.

51

348.

663.

210

060

134

9.46

0.1

1006

01

349.

160.

510

060

134

9.36

0.5

1006

03

2C

DS

.25

35

1.26

0.5

——

13

51

.26

1.0

10

060

13

51

.26

1.0

10

060

13

51

.26

1.0

10

060

33C

DS

.25.

CA

M34

2.36

3.7

——

132

6.96

4.0

40.5

619

.11

326.

964.

040

.56

19.1

132

8.56

6.5

42.7

619

.32

348.

162.

859

.56

19.1

234

8.16

2.8

59.5

619

.12

348.

063.

557

.36

19.3

34C

AB

339.

560.

61

339.

860.

610

060

133

9.86

0.7

1006

01

339.

760.

610

060

133

9.86

0.1

1006

035

CA

H(2

-com

p.)

332.

860.

41

325.

963.

349

.86

24.9

132

0.56

4.6

26.4

615

.51

322.

364.

943

.36

19.2

132

5.96

3.3

49.8

624

.92

338.

663.

150

.26

24.9

233

5.96

1.9

73.6

615

.52

336.

862.

656

.86

19.2

233

8.66

3.1

50.2

624

.9(3

-com

p.)

——

131

7.16

3.8

15.3

64.

71

317.

163.

815

.36

4.7

131

7.16

3.8

15.3

64.

72

333.

161.

460

.06

18.2

233

3.16

1.4

60.0

618

.22

333.

161.

460

.06

18.2

334

0.36

4.7

24.7

613

.53

340.

364.

724

.76

13.5

334

0.36

4.7

24.7

613

.536

CP

A(2

-com

p.)

331.

060.

31

327.

761.

677

.56

8.4

132

5.76

1.6

66.5

612

.81

326.

461.

870

.26

12.0

132

6.26

1.5

69.1

612

.42

347.

464.

422

.56

8.4

234

1.76

3.0

33.5

612

.82

343.

664.

229

.86

12.0

234

2.66

3.1

30.9

612

.4(3

-com

p.)

——

130

4.46

11.9

18.6

611

.31

304.

4611

.918

.66

11.3

130

4.46

11.9

18.6

611

.32

330.

761.

673

.26

12.7

233

0.76

1.6

73.2

612

.72

330.

761.

673

.26

12.7

335

1.56

2.9

8.26

1.7

335

1.56

2.9

8.26

1.7

335

1.56

2.9

8.26

1.7

37C

HT

333.

761.

31

329.

362.

257

.66

14.6

133

0.36

2.5

64.6

621

.21

330.

663.

464

.86

17.6

132

8.26

2.7

54.9

617

.02

341.

663.

342

.46

14.6

234

2.16

3.5

35.4

621

.22

342.

463.

635

.26

17.6

234

1.06

2.9

45.1

617

.03

8C

HT

.D3

53

39

.262

.41

32

8.8

60

.12

6.1

62

6.4

13

24

.26

12

.83

4.8

62

0.6

13

26

.16

9.6

32

.36

23

.21

32

4.2

61

2.8

34

.86

20

.62

34

2.5

61

.37

3.9

62

6.4

23

40

.76

2.5

65

.26

20

.62

34

1.6

62

.06

7.7

62

3.2

23

40

.76

2.5

65

.26

20

.63

9C

HT

.D6

0-8

03

39

.860

.11

32

7.8

61

.33

4.0

61

.91

32

8.9

62

.04

4.0

61

0.3

13

28

.56

1.8

35

.36

1.9

13

27

.86

1.3

34

.06

1.9

23

43

.76

1.6

66

.06

1.9

23

43

.96

1.1

56

.06

10

.32

34

4.3

61

.46

4.7

61

.92

34

3.7

61

.66

6.0

61

.940

CH

G(1

-com

p.)

331.

760.

21

332.

060.

510

060

133

1.96

0.4

1006

01

331.

960.

410

060

133

2.26

0.7

1006

0(2

-com

p.)

132

7.56

1.5

61.5

613

.91

327.

963.

150

.06

29.4

132

7.76

2.3

55.7

622

.81

327.

561.

358

.56

19.2

233

9.16

1.9

38.5

613

.92

338.

766.

550

.06

29.4

233

8.96

4.6

44.3

622

.82

339.

464.

041

.56

19.2

41

CH

G.D

35

33

3.26

0.5

13

22

.76

2.9

33

.36

0.2

13

16

.76

4.5

35

.96

11

.11

31

8.7

64

.73

0.7

63

.71

32

2.7

62

.93

3.3

–0

.22

33

6.2

63

.06

6.7

60

.12

33

5.6

60

.16

4.1

61

1.1

23

35

.86

0.4

69

.36

3.7

23

36

.26

3.0

66

.76

0.1

Biophysical Journal 81(3) 1710–1734

Page 11: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

42

CH

G.D

60

-70

33

9.26

0.3

13

36

.86

0.9

69

.66

9.3

13

36

.06

1.8

61

.06

28

.41

33

6.2

61

.56

4.9

62

1.0

13

36

.86

0.9

69

.66

9.3

23

47

.56

3.8

30

.46

9.3

23

45

.96

5.1

39

.06

28

.42

34

6.5

64

.33

5.1

62

1.0

23

47

.56

3.8

30

.46

9.3

43

CH

G.U

R3

49

.760

.51

34

8.4

63

.11

006

01

35

0.0

60

.41

006

01

34

9.4

60

.51

006

01

35

0.0

63

.21

006

044

CP

K33

3.56

0.4

132

3.96

2.8

34.1

66.

41

321.

362.

934

.96

3.7

132

1.96

2.8

34.0

64.

41

320.

963.

934

.86

5.4

233

8.96

0.9

65.9

66.

42

338.

760.

465

.16

3.7

233

8.76

0.5

66.0

64.

42

338.

960.

765

.26

5.4

45E

NH

(1-c

omp.

)33

2.66

0.1

133

2.36

0.1

1006

01

332.

560.

110

060

133

2.46

0.2

1006

01

332.

260.

210

060

(2-c

omp.

)1

327.

760.

847

.66

13.0

132

9.66

1.0

53.0

617

.81

329.

061.

344

.16

11.9

132

7.76

0.8

47.6

613

.02

336.

661.

452

.46

13.0

233

4.66

0.3

47.0

617

.82

335.

261.

355

.96

11.9

233

6.66

1.4

52.4

613

.046

FG

E34

5.86

0.8

133

0.76

7.6

23.5

614

.31

333.

062.

626

.76

7.9

133

2.26

4.2

27.4

69.

91

330.

767.

623

.56

14.3

235

1.26

3.7

76.5

614

.32

351.

362.

373

.36

7.9

235

1.36

2.4

72.6

69.

92

351.

263.

776

.56

14.3

47F

BN

331.

860.

61

321.

761.

345

.56

5.8

132

1.66

2.5

60.7

612

.01

321.

662.

155

.46

11.8

132

1.66

2.5

60.7

612

.02

340.

061.

764

.56

5.8

234

5.06

3.4

39.3

612

.02

343.

363.

844

.66

11.8

234

5.06

3.4

39.3

612

.048

FIL

337.

460.

71

326.

863.

726

.86

6.5

132

0.66

2.8

31.7

63.

51

322.

664.

229

.56

5.0

132

1.06

2.6

32.0

61.

32

341.

863.

373

.26

6.5

234

2.86

1.7

68.3

63.

52

342.

563.

270

.56

5.0

234

3.06

1.7

68.0

61.

349

GLG

350.

860.

11

349.

660.

410

060

135

0.86

1.0

1006

01

350.

460.

710

060

135

0.86

0.1

1006

050

GP

D33

4.96

0.8

132

7.46

1.7

57.9

613

.61

328.

763.

063

.96

17.0

132

8.66

2.7

59.4

614

.31

329.

362.

162

.86

15.2

234

5.56

3.2

42.1

613

.62

345.

263.

236

.16

17.0

234

5.86

4.0

40.6

614

.32

346.

363.

337

.26

15.2

51G

PS

342.

560.

11

341.

860.

210

060

134

2.56

1.0

1006

01

342.

261.

010

060

134

2.56

0.1

1006

05

2H

BA

.CM

34

9.46

0.5

13

24

.66

0.8

3.4

63

.11

33

0.3

63

.47

.96

1.9

13

27

.56

3.8

5.6

63

.41

32

4.6

60

.83

.46

3.1

23

50

.76

0.3

96

.66

3.1

23

51

.46

0.2

92

.16

1.9

23

51

.06

0.5

94

.46

3.4

23

50

.76

0.3

96

.66

3.1

53IG

G33

4.76

0.3

132

8.46

1.3

53.1

63.

71

325.

464.

029

.46

6.4

132

6.96

3.0

41.3

614

.31

328.

360.

946

.76

11.4

233

9.46

0.5

46.9

63.

72

336.

660.

470

.66

6.4

233

8.06

1.7

58.7

614

.32

338.

361.

853

.36

11.4

54

IGG

.UR

34

8.36

0.5

13

44

.06

3.2

51

.86

0.6

13

46

.66

0.5

55.8

610

.01

34

5.3

61

.85

3.8

62

.81

34

6.6

60

.55

5.8

61

0.0

23

54

.66

3.5

48

.26

0.6

23

51

.26

0.5

44

.26

9.0

23

52

.96

2.4

46

.26

2.8

23

51

.26

0.5

44

.26

9.0

55IO

P33

4.66

0.6

132

3.16

2.8

47.2

67.

21

327.

960.

571

.16

1.3

132

6.36

2.8

63.1

612

.71

327.

960.

571

.16

1.3

234

7.76

2.3

52.8

67.

22

359.

561.

529

.06

1.3

235

5.66

6.3

36.9

612

.72

359.

561.

529

.06

1.3

56LA

B32

5.96

0.2

132

2.46

1.1

74.9

63.

41

321.

963.

970

.16

18.1

132

2.16

2.8

76.2

66.

41

322.

461.

174

.96

3.4

233

8.36

3.8

25.1

63.

42

338.

063.

029

.96

18.1

233

8.16

6.8

23.8

66.

42

338.

363.

825

.16

3.4

57LA

H32

7.36

0.3

132

2.46

2.2

67.2

64.

91

324.

561.

082

.56

5.5

132

3.66

1.7

74.5

69.

51

322.

462.

267

.26

4.9

234

1.96

2.1

32.8

64.

92

350.

165.

817

.56

5.5

234

6.66

6.1

25.5

69.

52

341.

962.

132

.86

4.9

58LG

B.A

C32

3.36

0.3

——

133

2.36

0.3

1006

01

332.

360.

310

060

133

2.36

0.4

1006

059

LGB

.N33

2.16

0.5

——

133

2.26

0.1

1006

01

332.

260.

110

060

133

2.16

0.5

1006

060

LGB

.AL

333.

360.

5—

—1

333.

360.

310

060

133

3.36

0.2

1006

01

333.

360.

510

060

61LG

C.A

C33

7.56

0.2

132

3.26

6.9

29.0

628

.11

326.

161.

130

.56

5.3

132

5.46

3.1

29.6

618

.31

326.

161.

130

.56

5.3

234

7.56

7.7

71.0

628

.12

343.

660.

369

.56

5.3

234

3.36

0.7

70.4

618

.32

343.

660.

369

.56

5.3

62LG

C.N

329.

160.

81

322.

961.

155

.86

2.0

132

4.16

0.4

64.3

60.

11

323.

861.

160

.16

5.0

132

4.16

0.4

64.3

60.

1(2

-com

p.)

234

7.36

0.4

44.2

62.

02

349.

061.

135

.76

0.1

234

8.46

1.3

39.9

65.

02

349.

061.

135

.76

0.1

(3-c

omp.

)—

—1

323.

760.

454

.66

6.6

132

3.76

0.4

54.6

66.

61

323.

760.

454

.66

6.6

233

5.56

8.6

21.4

62.

82

335.

568.

621

.46

2.8

233

5.56

8.6

21.4

62.

83

353.

064.

024

.26

9.3

335

3.06

4.0

24.2

69.

33

353.

064.

024

.26

9.3

63LP

P(2

-com

p.)

344.

060.

31

326.

363.

422

.16

7.1

132

6.76

2.4

22.1

68.

41

327.

664.

724

.76

6.0

132

6.36

3.4

22.1

67.

12

351.

661.

977

.96

7.1

235

1.06

1.9

77.9

68.

42

351.

261.

875

.36

6.0

235

1.66

1.9

77.9

67.

1(3

-com

p.)

——

132

5.56

2.7

17.0

68.

91

325.

562.

717

.06

8.9

132

5.56

2.7

17.0

68.

92

339.

267.

224

.46

8.7

233

9.26

7.2

24.4

68.

72

339.

267.

224

.46

8.7

335

3.86

1.2

58.6

613

.53

353.

861.

258

.66

13.5

335

3.86

1.2

58.6

613

.564

LWG

335.

860.

41

325.

360.

438

.36

2.5

132

9.86

0.6

55.5

65.

01

328.

362.

448

.06

11.3

132

9.76

0.3

57.6

60.

8

Biophysical Journal 81(3) 1710–1734

Page 12: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

234

3.86

0.2

61.7

62.

52

345.

961.

344

.56

5.0

234

5.26

1.5

52.0

611

.32

346.

460.

642

.46

0.8

65LZ

C.A

C33

7.26

0.2

133

0.06

1.2

44.5

65.

71

329.

961.

445

.86

7.7

132

9.96

1.2

42.9

66.

81

330.

061.

244

.56

5.7

(2-c

omp.

)2

346.

460.

855

.56

5.7

234

5.66

12.2

54.2

67.

72

345.

861.

857

.16

6.8

234

6.46

0.8

55.5

65.

7(3

-com

p.)

——

131

7.46

0.3

8.76

2.8

131

7.46

0.3

8.76

2.8

131

7.46

0.3

8.76

2.8

233

3.66

1.7

41.4

60.

32

333.

661.

741

.46

0.3

233

3.66

1.7

41.4

60.

33

345.

761.

949

.96

2.4

334

5.76

1.9

49.9

62.

43

345.

761.

949

.96

2.4

66LZ

C.N

338.

360.

2—

—1

313.

960.

55.

061.

01

313.

960.

55.

061.

01

313.

960.

55.

061.

02

334.

860.

567

.56

5.0

233

4.86

0.5

67.5

65.

02

334.

860.

567

.56

5.0

334

3.06

0.5

27.5

68.

03

343.

060.

527

.56

8.0

334

3.06

0.5

27.5

68.

06

7L

ZC

.UR

34

5.26

3.7

13

33

.26

4.5

30

.26

34

.11

34

0.6

66

.65

0.6

62

.61

33

7.6

66

.64

0.0

62

2.7

13

42

.36

8.3

53

.16

1.8

23

50

.16

2.1

69

.86

34

.12

34

9.0

61

.14

9.4

62

.62

34

9.4

61

.36

0.0

62

2.7

23

50

.76

1.3

46

.96

1.8

68M

SP

349.

860.

21

349.

660.

110

060

135

0.06

0.2

1006

01

349.

060.

210

060

134

9.96

0.2

1006

069

MLT

.MO

N34

9.86

0.5

——

134

9.26

0.5

1006

01

349.

260.

510

060

134

9.86

0.5

1006

070

MLT

.TE

TR

336.

961.

71

331.

364.

956

.66

21.9

132

9.46

4.1

38.5

611

.11

330.

064.

147

.36

17.9

132

9.56

4.5

48.7

613

.92

348.

066.

543

.46

21.9

234

2.36

1.8

61.5

611

.12

344.

264.

552

.76

17.9

234

3.96

3.3

51.3

613

.971

MLT

.CA

M33

8.3

60.

5—

—1

332.

361.

971

.56

12.2

133

2.36

1.9

71.5

612

.21

330.

960.

562

.86

2.0

235

2.86

3.3

28.5

612

.22

352.

863.

328

.56

12.2

235

0.56

0.5

37.2

62.

072

MLT

.LS

332.

260.

51

327.

362.

965

.26

0.1

132

3.26

3.0

50.0

624

.71

324.

563.

242

.56

31.2

132

4.56

3.2

42.5

631

.22

343.

563.

034

.86

0.1

233

5.76

0.2

50.0

624

.72

338.

364.

557

.56

31.2

233

8.36

4.5

57.5

631

.273

HM

M33

7.56

0.5

132

4.06

5.2

17.6

62.

81

323.

660.

513

.06

2.0

132

3.86

0.3

15.2

63.

11

323.

660.

513

.06

2.0

234

0.16

3.1

82.4

60.

62

339.

360.

587

.06

3.0

234

1.16

2.5

84.8

63.

12

339.

360.

587

.06

3.0

74LM

M33

9.4

60.

51

337.

463.

110

060

133

9.46

0.6

1006

01

339.

160.

910

060

133

9.46

0.6

1006

075

MO

N(1

-com

p.)

340.

760.

41

341.

160.

510

060

134

1.16

0.5

1006

01

340.

261.

310

060

134

0.36

1.5

1006

0(2

-com

p.)

133

1.06

3.4

28.3

610

.91

323.

967.

122

.76

8.2

132

5.66

7.2

22.9

68.

81

327.

566.

625

.96

8.6

234

4.86

1.1

71.7

610

.92

344.

161.

377

.36

8.2

234

4.36

1.3

77.1

68.

82

344.

161.

574

.16

8.6

76M

GB

.AP

O33

6.46

0.5

131

9.16

7.8

22.5

619

.21

325.

663.

228

.36

8.6

132

3.46

5.4

24.4

613

.01

325.

363.

722

.56

19.2

234

0.56

2.7

77.5

619

.22

340.

260.

671

.76

8.6

234

0.36

1.3

75.6

613

.02

340.

360.

777

.56

19.2

77M

SS

336.

960.

41

327.

069.

641

.16

28.2

132

8.66

4.8

24.4

626

.21

328.

066.

328

.06

23.1

132

3.46

6.6

12.3

62.

72

342.

265.

559

.96

28.2

233

9.46

1.7

75.6

626

.22

340.

562.

772

.06

23.1

233

8.66

0.6

87.7

62.

77

8M

SS

.UR

34

5.16

0.5

13

38

.26

3.3

31

.96

1.1

13

40

.36

0.5

38

.76

6.0

13

40

.56

2.5

35

.36

4.8

13

38

.26

3.3

31

.96

1.1

23

48

.76

3.3

68

.16

0.6

23

48

.46

0.1

61

.36

5.0

23

48

.56

0.2

64

.86

4.8

23

48

.76

3.3

68

.16

0.6

79M

S1

335.

760.

4—

—1

318.

861.

320

.76

3.0

131

8.86

1.3

20.7

63.

01

318.

861.

320

.76

3.0

233

1.86

0.9

23.0

60.

52

331.

860.

923

.06

0.5

233

1.86

0.9

23.0

60.

53

342.

560.

556

.36

2.5

334

2.56

0.5

56.3

62.

53

342.

560.

556

.36

2.5

80M

S1.

AT

P33

5.56

0.3

——

131

7.46

1.8

13.8

63.

01

317.

461.

813

.86

3.0

131

7.46

1.8

13.8

63.

02

330.

362.

428

.86

5.7

233

0.36

2.4

28.8

65.

72

330.

362.

428

.86

5.7

333

9.26

0.5

57.5

68.

73

339.

260.

557

.56

8.7

333

9.26

0.5

57.5

68.

781

NO

1(1

-com

p.)

346.

160.

21

346.

660.

410

060

134

6.56

0.6

1006

01

346.

560.

610

060

134

6.66

0.4

1006

0(2

-com

p.)

132

9.16

4.4

4.96

1.3

133

5.66

6.8

28.1

620

.11

332.

866.

519

.56

20.3

132

9.16

4.4

4.96

1.3

234

7.46

0.3

95.1

61.

32

350.

162.

371

.96

20.1

234

8.96

2.2

80.5

620

.32

347.

460.

395

.16

1.3

82N

O2

344.

560.

21

344.

163.

110

060

134

4.56

0.3

1006

01

344.

460.

310

060

134

4.46

0.2

1006

083

NS

T33

6.06

0.1

133

0.96

0.8

45.7

65.

71

327.

067.

241

.76

44.1

132

8.36

6.1

33.3

614

.21

330.

960.

845

.76

5.7

234

0.76

0.4

54.9

65.

72

339.

461.

058

.36

44.1

233

9.86

1.0

66.7

614

.22

340.

760.

454

.96

5.7

84N

DP

K.A

.834

0.56

0.4

134

0.26

0.4

1006

01

340.

460.

410

060

134

0.46

0.4

1006

01

340.

460.

310

060

85N

DP

K.A

.5.L

IS33

7.36

0.8

——

132

6.46

4.7

25.9

622

.61

326.

464.

725

.96

22.6

132

7.06

5.0

17.9

611

.72

340.

062.

174

.16

22.6

234

0.06

2.1

74.1

622

.62

340.

062.

382

.16

11.7

Biophysical Journal 81(3) 1710–1734

Page 13: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

86N

DP

K.A

.5.H

IS33

6.56

0.3

132

9.16

0.4

39.4

61.

71

323.

864.

520

.06

10.9

132

5.56

4.4

26.5

613

.01

325.

164.

524

.36

12.6

234

2.26

0.6

60.6

61.

72

339.

061.

380

.06

10.9

234

0.16

1.9

73.5

613

.02

339.

061.

875

.76

12.6

87N

DP

K.B

.833

5.56

0.3

133

5.36

0.8

1006

01

335.

560.

310

060

133

5.46

0.3

1006

01

335.

460.

510

060

88N

DP

K.B

.AC

334.

260.

8—

—1

334.

760.

710

060

133

4.76

0.7

1006

01

334.

461.

110

060

(1-c

omp.

)(2

-com

p.)

——

133

0.06

0.7

78.9

66.

11

330.

060.

778

.96

6.1

133

0.06

0.7

78.9

66.

12

353.

964.

921

.16

6.1

235

3.96

4.9

21.1

66.

12

353.

964.

921

.16

6.1

89O

VH

.LIS

334.

460.

51

325.

862.

617

.66

0.2

132

7.56

0.2

23.0

60.

41

326.

961.

020

.46

4.0

132

5.86

2.6

17.6

60.

22

336.

962.

782

.46

0.1

233

7.06

0.4

77.0

60.

42

336.

960.

379

.66

4.0

233

6.96

2.7

82.4

60.

190

OV

H.H

IS33

4.96

0.1

133

5.16

0.2

1006

01

335.

260.

210

060

133

5.16

0.2

1006

01

334.

960.

110

060

91P

AP

.AC

340.

261.

91

326.

868.

343

.16

16.7

132

3.56

5.4

44.9

617

.81

324.

666.

439

.76

15.3

132

3.86

7.3

37.7

613

.4(2

-com

p.)

234

9.76

3.2

56.9

616

.72

349.

863.

755

.16

17.8

234

9.86

3.4

60.3

615

.32

348.

563.

262

.36

13.4

(3-c

omp.

)—

—1

304.

266.

614

.06

5.4

130

4.26

6.6

14.0

65.

41

304.

266.

614

.06

5.4

233

1.96

5.5

39.5

613

.52

331.

965.

539

.56

13.5

233

1.96

5.5

39.5

613

.53

351.

163.

746

.56

17.5

335

1.16

3.7

46.5

617

.53

351.

163.

746

.56

17.5

92P

AP

.N34

3.36

0.8

132

4.86

4.0

19.3

68.

31

327.

464.

429

.56

13.7

132

6.56

4.2

20.3

66.

41

324.

864.

019

.36

8.3

(2-c

omp.

)2

348.

561.

380

.76

8.3

234

9.16

0.9

70.5

613

.72

348.

961.

079

.76

6.4

234

8.56

1.3

80.7

68.

3(3

-com

p.)

——

131

7.76

6.9

17.5

65.

91

317.

766.

917

.56

5.9

131

7.76

6.9

17.5

65.

92

341.

063.

920

.16

13.0

234

1.06

3.9

20.1

613

.02

341.

063.

920

.16

13.0

334

9.66

4.0

62.4

615

.73

349.

664.

062

.46

15.7

334

9.66

4.0

62.4

615

.793

PA

P.A

L34

8.26

0.6

134

9.06

1.2

1006

01

348.

761.

010

060

134

8.46

1.0

1006

01

348.

561.

210

060

(1-c

omp.

)(2

-com

p.)

133

3.66

8.8

10.3

68.

51

337.

766.

823

.26

28.6

133

6.16

7.3

21.7

626

.31

333.

668.

810

.36

8.5

234

9.66

0.4

89.7

68.

52

350.

660.

676

.86

28.6

235

0.26

0.7

78.3

626

.32

349.

660.

489

.76

8.5

94

PA

P.U

R3

44

.060

.51

31

6.8

64

.31

8.4

60

.71

32

0.7

63

.52

1.8

65

.81

31

9.4

63

.31

8.1

60

.51

31

8.2

60

.51

7.7

61

.02

34

6.5

63

.18

1.6

60

.72

34

6.1

60

.17

8.2

65

.82

34

6.2

60

.38

1.9

60

.52

34

6.0

60

.58

2.3

61

.095

PA

C.A

C34

1.56

0.5

——

131

0.16

0.6

8.66

3.5

131

0.16

0.6

8.66

3.5

130

9.66

2.0

6.16

3.0

(2-c

omp.

)2

342.

660.

491

.46

3.5

234

2.66

0.4

91.4

63.

52

342.

362.

093

.96

3.0

(3-c

omp.

)—

—1

309.

662.

06.

163.

01

309.

662.

06.

163.

01

309.

662.

06.

163.

02

338.

860.

566

.76

3.0

233

8.86

0.5

66.7

63.

02

338.

860.

566

.76

3.0

335

0.86

0.5

27.2

64.

03

350.

860.

527

.26

4.0

335

0.86

0.5

27.2

64.

096

PA

C.A

PO

348.

060.

5—

—1

308.

9612

.017

.06

6.2

130

8.96

12.0

17.0

66.

21

300.

464.

022

.16

7.8

234

9.46

0.6

83.0

66.

22

349.

460.

683

.06

6.2

234

9.06

0.5

77.8

61.

097

PA

C.M

G33

1.16

0.5

——

131

8.16

0.5

55.3

62.

01

318.

160.

555

.36

2.0

131

8.16

0.5

55.3

62.

02

341.

160.

544

.76

2.0

234

1.16

0.5

44.7

62.

02

341.

160.

544

.76

2.0

98P

AC

.CA

325.

860.

5—

—1

326.

560.

710

060

132

6.56

0.7

1006

01

326.

560.

710

060

99P

AM

.AP

O33

6.66

0.5

132

6.96

2.9

43.9

60.

11

327.

762.

654

.96

17.5

132

7.46

1.9

43.2

61.

01

326.

962.

943

.96

0.1

(2-c

omp.

)2

345.

763.

156

.16

0.1

234

1.76

3.4

45.1

617

.52

346.

662.

556

.86

1.0

234

5.76

3.1

56.1

60.

1(3

-com

p.)

——

131

0.66

0.5

8.86

1.0

131

0.66

0.5

8.86

1.0

131

0.66

0.5

8.86

1.0

233

2.46

0.5

58.4

61.

02

332.

460.

558

.46

1.0

233

2.46

0.5

58.4

61.

03

349.

560.

532

.86

3.0

334

9.56

0.5

32.8

63.

03

349.

560.

532

.86

3.0

100

PA

M(2

-com

p.)

336.

460.

51

328.

564.

130

.16

1.3

131

8.96

1.5

36.3

63.

31

322.

165.

632

.16

2.8

131

7.96

0.5

34.0

62.

02

340.

463.

469

.96

0.6

234

1.76

0.2

63.7

63.

32

341.

260.

767

.96

2.8

234

1.56

0.5

66.0

62.

0(3

-com

p.)

——

131

7.26

0.5

32.1

63.

01

317.

260.

532

.16

3.0

131

7.26

0.5

32.1

63.

02

333.

260.

56.

562.

02

333.

260.

56.

562.

02

333.

260.

56.

562.

03

341.

860.

561

.46

7.0

334

1.86

0.5

61.4

67.

03

341.

860.

561

.46

7.0

Biophysical Journal 81(3) 1710–1734

Page 14: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

101

PM

T.A

PO

349.

960.

5—

—1

349.

860.

110

060

134

9.86

0.1

1006

01

349.

860.

510

060

(1-c

omp.

)(2

-com

p.)

——

133

1.26

0.5

9.56

2.0

133

1.26

0.5

9.56

2.0

133

1.26

0.5

9.56

2.0

235

1.76

0.5

90.5

62.

02

351.

760.

590

.56

2.0

235

1.76

0.5

90.5

62.

010

2P

MT

.CA

324.

860.

5—

—1

324.

461.

110

060

132

4.56

0.8

1006

01

324.

860.

510

060

(1-c

omp.

)—

—1

313.

061.

518

.36

2.0

131

3.06

1.5

18.3

62.

01

313.

061.

518

.36

2.0

(2-c

omp.

)2

326.

060.

581

.76

5.0

232

6.06

0.5

81.7

65.

02

326.

060.

581

.76

5.0

103

PM

T.M

G32

.26

0.5

——

133

2.06

0.2

1006

01

332.

060.

210

060

133

1.86

0.5

1006

0(1

-com

p.)

(2-c

omp.

)—

—1

325.

160.

574

.16

1.0

132

5.16

0.5

74.1

61.

01

325.

160.

574

.16

1.0

235

1.36

0.5

25.9

61.

02

351.

360.

525

.96

1.0

235

1.36

0.5

25.9

61.

010

4P

AN

.AP

O34

8.66

0.5

——

132

1.46

2.5

6.56

0.8

132

1.46

2.5

6.56

0.8

132

3.16

0.5

7.16

1.0

234

9.96

0.2

93.5

60.

82

349.

960.

293

.56

0.8

235

0.06

0.5

92.9

61.

010

5P

AN

.CA

324.

760.

5—

—1

324.

760.

110

060

132

4.76

0.1

1006

01

324.

760.

510

060

106

PA

N.M

G33

2.66

0.5

——

131

7.06

1.8

42.4

63.

51

317.

061.

842

.46

3.5

131

8.26

0.5

44.8

62.

02

339.

260.

857

.66

3.5

233

9.26

0.8

57.6

63.

52

339.

760.

855

.26

2.0

107

PE

B.1

-334

0.26

0.4

133

4.46

1.4

52.0

613

.81

332.

064.

944

.06

21.9

133

2.86

4.1

38.3

617

.51

330.

866.

241

.96

23.0

(2-c

omp.

)2

347.

462.

448

.06

13.8

234

3.96

1.5

56.0

621

.92

345.

162.

461

.76

17.5

234

5.96

3.4

58.1

623

.0(3

-com

p.)

——

131

9.46

4.2

12.1

66.

81

319.

464.

212

.16

6.8

131

9.46

4.2

12.1

66.

82

340.

760.

851

.46

12.6

234

0.76

0.8

51.4

612

.62

340.

760.

851

.46

12.6

334

4.36

2.2

36.5

66.

63

344.

362.

236

.56

6.6

334

4.36

2.2

36.5

66.

610

8P

EB

.4-5

342.

560.

81

327.

567.

022

.06

15.6

132

7.46

4.6

20.6

66.

51

327.

465.

219

.06

11.0

132

5.56

6.0

15.7

67.

2(2

-com

p.)

234

7.16

4.8

78.0

615

.62

345.

762.

179

.46

6.5

234

6.26

3.2

81.0

611

.02

345.

161.

684

.36

7.2

(3-c

omp.

)—

—1

317.

564.

112

.06

6.0

131

7.56

4.1

12.0

66.

01

317.

564.

112

.06

6.0

233

8.76

4.6

14.8

66.

02

338.

764.

614

.86

6.0

233

8.76

4.6

14.8

66.

03

345.

962.

973

.26

2.1

334

5.96

2.9

73.2

62.

13

345.

962.

973

.26

2.1

10

9P

EB

.6-9

34

2.56

0.5

13

43

.46

0.4

10

060

13

42

.96

0.6

10

060

13

43

.16

0.6

10

060

13

42

.36

0.4

10

060

(1-c

om

p.)

(2-c

om

p.)

13

37

.262

.34

4.2

61

0.4

13

35

.26

3.3

36

.26

11

.41

33

6.2

62

.74

2.4

61

0.3

13

36

.56

2.8

48

.56

10

.22

34

8.6

62

.05

5.8

61

0.4

23

46

.96

0.6

63

.86

11

.42

34

7.6

61

.55

7.6

61

0.3

23

49

.76

1.0

51

.56

10

.21

10

PE

B.U

R3

48

.160

.51

32

6.2

63

.11

0.3

60

.51

32

7.6

67

.51

2.5

66

.91

32

7.1

65

.41

3.9

65

.01

32

6.2

63

.11

0.3

60

.52

35

0.0

63

.18

9.7

60

.12

34

9.9

61

.18

7.5

66

.92

34

9.9

60

.88

6.1

65

.02

35

0.0

63

.18

9.7

60

.111

1P

HA

327.

360.

51

320.

260.

761

.06

5.2

132

3.36

1.5

81.1

64.

71

322.

562.

074

.86

11.7

132

2.86

2.1

76.2

613

.52

337.

562.

339

.06

5.2

234

6.56

2.5

18.9

64.

72

345.

267.

125

.26

11.7

234

5.86

9.9

23.8

613

.511

2P

GK

335.

860.

21

323.

763.

241

.06

3.1

132

3.76

0.4

53.8

60.

11

323.

762.

047

.26

7.5

132

3.06

0.8

48.7

68.

72

344.

661.

759

.06

3.1

234

8.16

1.5

46.2

60.

12

346.

362.

452

.86

7.5

234

6.66

1.1

51.3

68.

711

3P

LB34

4.36

0.5

133

1.86

2.4

21.6

617

.71

330.

064.

835

.56

4.7

133

0.56

4.1

30.4

612

.21

331.

764.

536

.76

2.7

234

9.06

3.7

79.4

617

.72

351.

561.

565

.56

4.7

235

0.86

2.3

69.6

612

.22

352.

361.

563

.36

2.7

114

PLS

343.

560.

11

323.

7613

.728

.76

22.6

132

5.56

4.7

21.2

68.

91

324.

967.

224

.76

14.2

132

3.76

8.5

27.3

616

.12

350.

665.

671

.36

22.6

234

8.36

1.5

78.8

68.

92

349.

063.

075

.36

14.2

234

9.46

3.6

72.7

616

.111

5P

AO

.LIS

337.

160.

4—

—1

320.

164.

735

.36

10.6

132

0.16

4.7

35.3

610

.61

322.

462.

540

.96

7.9

234

3.46

2.9

64.7

610

.62

343.

462.

964

.76

10.6

234

4.76

2.8

59.1

67.

911

6P

AO

.HIS

344.

060.

21

329.

062.

321

.96

6.9

133

0.36

2.8

28.3

611

.91

329.

962.

529

.76

9.8

133

1.46

1.1

26.9

610

.02

349.

961.

278

.16

6.9

235

0.86

2.3

71.8

611

.92

350.

561.

970

.36

9.8

235

1.66

1.3

73.1

610

.01

17

PA

O.G

DN

34

6.56

0.5

——

13

46

.66

0.2

10

060

13

46

.66

0.2

10

060

13

46

.56

0.5

10

060

Biophysical Journal 81(3) 1710–1734

Page 15: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

118

PLC

(1-c

omp.

)34

3.36

0.3

——

134

2.96

0.6

1006

01

342.

960.

610

060

134

3.06

0.2

1006

0(2

-com

p.)

——

132

7.66

2.6

8.46

0.4

132

7.66

2.6

8.46

0.4

132

7.66

2.6

8.46

0.4

234

4.36

0.1

91.6

60.

42

344.

360.

191

.66

0.4

234

4.36

0.1

91.6

60.

411

9P

RL*

(1-c

omp.

)33

6.96

0.3

——

133

7.36

0.4

1006

01

337.

360.

410

060

133

6.96

0.4

1006

0(2

-com

p.)

——

133

4.06

1.2

79.7

69.

21

334.

061.

279

.76

9.2

133

3.66

0.5

83.2

62.

02

352.

264.

220

.26

9.2

235

2.26

4.2

20.2

69.

22

354.

861.

516

.86

4.0

120

PC

E33

5.36

0.1

132

3.56

1.4

46.3

62.

21

329.

960.

075

.26

0.7

132

6.76

3.8

60.7

616

.81

327.

464.

365

.06

17.6

234

8.46

0.5

53.7

62.

22

355.

660.

124

.86

0.7

235

7.06

9.9

39.3

616

.82

359.

7610

.135

.06

17.7

121

PR

H.L

IS34

5.06

1.0

134

4.96

0.5

1006

01

345.

260.

910

060

134

5.06

0.9

1006

01

345.

460.

710

060

122

PR

H.H

IS34

2.86

0.7

132

8.86

3.5

23.3

63.

21

332.

261.

739

.16

3.7

133

1.16

2.7

30.8

69.

01

328.

863.

523

.36

3.2

234

8.56

1.9

76.7

63.

22

350.

761.

660

.96

3.7

234

9.96

1.9

69.2

69.

02

348.

561.

976

.76

3.2

123

PR

H.P

ST

344.

361.

11

344.

460.

510

060

134

4.66

1.1

1006

01

344.

6,1.

010

060

134

4.96

0.9

1006

012

4P

RS

333.

960.

11

333.

060.

910

060

133

3.96

0.2

1006

01

333.

660.

610

060

133

3.96

0.1

1006

012

5P

RK

332.

960.

61

323.

762.

933

.96

8.4

132

8.06

1.6

72.5

69.

81

326.

463.

058

.46

21.4

132

6.96

2.9

59.3

622

.22

337.

861.

466

.16

8.4

234

7.16

3.7

27.5

69.

82

343.

765.

541

.66

21.4

234

4.26

6.5

40.7

622

.212

6P

YK

338.

761.

31

328.

265.

432

.06

12.7

133

1.26

3.8

44.9

614

.41

330.

564.

533

.06

10.9

132

8.26

5.4

32.0

612

.72

344.

564.

168

.06

12.7

234

6.06

2.3

55.1

614

.42

345.

563.

067

.06

10.9

234

4.56

4.1

68.0

612

.712

7P

YK

.PE

G33

9.96

0.5

132

1.16

2.9

25.2

60.

11

325.

661.

845

.36

8.6

132

4.16

2.9

32.2

69.

91

321.

162.

925

.26

0.1

234

6.26

3.1

74.8

60.

02

350.

061.

754

.76

8.6

234

8.76

2.5

67.8

69.

92

346.

263.

174

.86

0.0

128

RE

C.A

PO

*32

9.96

0.5

——

131

9.46

0.5

51.3

61.

01

319.

460.

551

.36

1.0

131

9.46

0.5

51.3

61.

02

336.

760.

548

.76

3.0

233

6.76

0.5

48.7

63.

02

336.

760.

548

.76

3.0

129

RE

C.C

A*

336.

660.

5—

—1

326.

060.

550

.66

1.0

132

6.06

0.5

50.6

61.

01

326.

060.

550

.66

1.0

234

5.26

0.5

49.4

62.

02

345.

260.

549

.46

2.0

234

5.26

0.5

49.4

62.

013

0R

CG

339.

160.

71

325.

663.

333

.56

6.1

133

2.36

2.5

64.2

616

.51

330.

064.

253

.86

22.6

133

0.56

3.8

65.0

618

.32

346.

862.

266

.56

6.1

235

1.86

1.1

35.8

616

.52

348.

863.

246

.26

22.6

235

9.26

12.9

35.0

618

.313

1R

NC

(1-c

omp.

)32

7.46

0.4

132

7.56

0.6

1006

01

327.

560.

710

060

132

7.46

0.6

1006

01

327.

460.

610

060

(2-c

omp.

)1

324.

561.

669

.96

16.2

132

5.86

1.6

74.7

618

.51

325.

361.

764

.56

21.9

132

3.76

3.1

73.4

616

.12

335.

163.

530

.16

16.2

233

4.06

4.3

25.3

618

.52

334.

663.

935

.56

21.9

233

5.16

3.6

26.6

616

.113

2R

NI

334.

660.

51

318.

662.

928

.86

0.1

132

8.16

0.6

67.1

66.

31

324.

965.

550

.26

30.2

132

8.56

0.5

71.5

61.

02

342.

063.

071

.26

0.1

235

7.66

1.7

32.9

66.

32

352.

469.

149

.86

30.2

235

8.86

0.5

28.5

61.

013

3R

NT

325.

660.

61

325.

161.

010

060

132

4.66

1.9

1006

01

324.

561.

810

060

132

5.16

0.8

1006

013

4R

S4

(2-c

omp.

)33

6.16

0.1

133

0.06

1.8

57.1

64.

21

329.

660.

159

.16

4.6

132

9.86

1.0

58.1

63.

81

330.

061.

857

.16

4.2

234

5.16

0.1

42.9

64.

22

345.

261.

640

.96

4.6

234

5.16

0.9

41.9

63.

82

345.

160.

142

.96

4.2

(3-c

omp.

)—

—1

324.

660.

718

.56

2.5

132

4.66

0.7

18.5

62.

51

324.

660.

718

.56

2.5

233

5.06

0.8

61.6

611

.12

335.

060.

861

.66

11.1

233

5.06

0.8

61.6

611

.13

350.

863.

919

.96

8.7

335

0.86

3.9

19.9

68.

73

350.

863.

919

.96

8.7

135

RS

734

1.96

0.7

132

9.66

3.4

33.1

611

.91

324.

965.

326

.66

7.4

132

6.56

5.1

31.2

68.

01

329.

663.

431

.86

10.1

235

1.16

2.8

66.9

611

.92

349.

061.

673

.46

7.4

234

9.76

2.1

69.8

68.

02

351.

162.

868

.26

10.1

136

RIP

(1-c

omp.

)34

0.66

0.5

134

1.86

0.8

1006

01

342.

761.

910

060

134

2.06

1.5

1006

01

341.

961.

210

060

137

RC

A(2

-com

p.)

330.

561.

41

325.

863.

056

.46

20.8

132

6.96

1.1

83.1

66.

01

326.

261.

767

.66

18.6

132

4.96

1.8

62.7

621

.22

343.

764.

843

.66

20.8

235

3.46

6.4

16.9

66.

02

351.

267.

132

.46

18.6

234

5.56

4.9

37.3

621

.2(3

-com

p.)

——

132

4.06

3.3

44.7

625

.91

324.

063.

344

.76

25.9

132

4.06

3.3

44.7

625

.92

330.

361.

743

.96

23.7

233

0.36

1.7

43.9

623

.72

330.

361.

743

.96

23.7

335

9.06

3.8

11.4

63.

93

359.

063.

811

.46

3.9

335

9.06

3.8

11.4

63.

913

8R

CB

.433

6.16

1.3

133

1.76

1.9

48.9

612

.71

331.

561.

463

.76

11.0

133

1.66

1.4

60.3

614

.81

330.

161.

761

.06

12.0

234

5.96

3.2

51.1

612

.72

351.

064.

536

.36

11.0

234

8.36

6.5

39.7

614

.82

349.

765.

039

.06

12.0

Biophysical Journal 81(3) 1710–1734

Page 16: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

No.

Pro

tein

Cod

el

mto

tal(

nm)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

139

RC

B.4

.LA

C33

0.96

0.9

132

6.16

0.4

54.9

610

.51

326.

360.

874

.76

6.2

132

6.26

0.5

64.8

613

.41

326.

060.

477

.86

1.7

234

0.66

7.3

45.1

610

.52

349.

761.

325

.36

6.2

234

5.16

6.8

39.2

613

.42

348.

263.

522

.26

1.7

140

RC

B.5

-733

1.46

0.9

132

2.86

3.7

41.7

618

.31

326.

660.

962

.56

10.4

132

5.56

2.6

55.3

619

.11

324.

263.

258

.76

15.2

(2-c

omp.

)2

343.

166.

258

.36

18.3

234

4.96

3.2

37.5

610

.42

343.

863.

844

.76

19.1

234

4.56

4.6

41.3

615

.2(3

-com

p.)

——

132

7.06

1.0

54.9

611

.21

327.

061.

054

.96

11.2

132

7.06

1.0

54.9

611

.22

333.

761.

729

.06

11.1

233

3.76

1.7

29.0

611

.12

333.

761.

729

.06

11.1

335

6.06

2.7

16.2

61.

93

356.

062.

716

.26

1.9

335

6.06

2.7

16.2

61.

914

1R

CC

331.

461.

21

320.

562.

940

.66

11.4

132

7.26

1.1

67.8

612

.31

324.

964.

058

.36

20.1

132

4.16

3.7

58.4

620

.52

339.

863.

359

.46

11.4

234

6.06

5.0

32.2

612

.32

342.

867.

541

.76

20.1

234

5.66

6.8

41.6

620

.514

2S

TP

333.

560.

5—

—1

335.

661.

010

060

133

5.66

1.0

1006

01

335.

060.

510

060

143

SP

C(2

-com

p.)

341.

760.

11

324.

960.

413

.46

3.0

132

6.36

7.7

33.3

626

.91

326.

266.

018

.36

8.8

132

5.66

1.3

18.3

68.

82

344.

760.

186

.66

3.0

234

5.56

2.0

66.7

626

.92

345.

261.

581

.76

8.8

234

5.46

1.2

81.7

68.

8(3

-com

p.)

——

132

2.06

6.0

352.

262.

81

322.

066.

035

2.26

2.8

132

2.06

6.0

352.

262.

82

342.

163.

658

.66

14.7

234

2.16

3.6

58.6

614

.72

342.

163.

658

.66

14.7

335

2.26

2.8

24.4

60.

93

352.

262.

824

.46

0.9

335

2.26

2.8

24.4

60.

914

4B

PN

345.

760.

71

346.

260.

610

060

134

5.76

0.7

1006

01

346.

260.

710

060

134

6.26

0.6

1006

014

5S

LC35

0.06

2.0

131

5.86

8.6

17.5

65.

81

319.

864.

922

.06

4.6

131

8.46

6.5

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81

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01

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4.56

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p.)

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VT

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01

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310

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616

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325.

261.

664

.06

10.6

Biophysical Journal 81(3) 1710–1734

Page 17: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

obtained with two decomposition algorithms, SIMS (375components) and PHREQ (249 components), respectively.PanelsC and D reflect the results obtained using twoschemes of averaging (“Average” and “Mean Best Fit” inTable 2), respectively. All four histograms have the well-expressed deep minimum at 335–337 nm as a commonfeature. This feature seems to clearly demonstrate the exis-tence of a statistical discreteness of at least two large spec-tral classes of tryptophan residues in native proteins. More-over, panelsA–D demonstrate reproducible dips at 3296 1and 3476 1 nm, which may also mark the frontiers betweenoverlapping discrete classes of tryptophans in proteins.

However, the canonical form of histogram, constructedfrom columns, cannot reflect the accuracy of calculatedmaximum position values and of relative contributions ofcomponents in the whole emission spectrum. Therefore, weconstructed a new kind of histogram accounting for thesefactors (Figs. 3-5). In such histograms, each element (theindividual ith spectral component) is represented by thelittle Gauss distributiony(lm(i)) with maximum atlm(i),s(i) equal to the standard (root-mean-square one) error ofthe meanlm(i) value (see Table 2), and the maximal am-plitude of Gauss curves proportional to the relative contri-bution S(i) of the ith component to the total protein emis-sion. Such a histogram is a sum of these elements:

y~lm! 5 a~i!OiHS~i!

s~i!expFl 2 l~i!

s~i! G2JHere, S(i) is expressed in decimal fractions anda(i) is amultiplier equal to 1 if only one decomposition result isselected for a protein spectrum, or to 0.5 in the cases whentwo solutions with similar functional values are taken.

In such a representation, the histograms of Fig. 2 acquirethe view seen in Fig. 3 (curves N). In this case, theS(i)values are taken to be 1, ands 5 1.0 for every spectralcomponent. One can see that the main features of thecanonical histograms are retained in the new, Gauss-curve-based representation. Besides the histograms for compo-nents of spectra of native proteins, Fig. 3 also contains thecurves obtained for proteins denatured under various influ-ences (see Table 2). The shorter-wavelength “continua”reflect the fact that denaturation is not completed in somecases and the emission spectra possess components belong-ing to the retained populations of native and/or partly un-folded molecules. However, the highest peak at;350 nmand peaks or shoulders at 340–347 nm are due to thecomponents belonging to tryptophans in proteins denaturedby urea or guanidinium chloride and by dioxane, pH andother less unfolding denaturing factors. It is interesting thatthe histograms for denatured proteins also have the minimaat 335–340 nm, which position coincides with the deepminima of histograms for native proteins.

We constructed two more Gauss-curve-based histo-grams with values ofs(i) and S(i) varying according toN

o.P

rote

inC

ode

lm

tota

l(nm

)

PH

RE

QS

IMS

Ave

rage

Mea

nB

est

Fit

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

No.

lm

(i)

(nm

)S(

i)(%

)N

o.l

m(i

)(n

m)

S(i)

(%)

(2-c

omp.

)2

341.

967.

354

.96

16.6

234

5.96

4.4

40.5

610

.72

344.

665.

645

.46

16.5

234

7.66

4.2

36.0

610

.6(3

-com

p.)

——

132

4.16

2.1

43.2

621

.41

324.

162.

143

.26

21.4

132

4.16

2.1

43.2

621

.42

333.

766.

434

.86

15.2

233

3.76

6.4

34.8

615

.22

333.

766.

434

.86

15.2

335

0.86

3.6

22.0

67.

63

350.

863.

622

.06

7.6

335

0.86

3.6

22.0

67.

616

2aV

SC

.MO

D34

1.76

0.2

132

8.26

2.3

25.7

66.

91

330.

161.

232

.26

6.1

132

9.56

1.7

31.4

67.

81

328.

262.

325

.76

6.9

(2-c

omp.

)2

350.

760.

574

.36

6.9

235

5.16

2.9

67.8

66.

12

353.

363.

268

.66

7.8

235

0.76

0.5

74.3

66.

91

62

bV

SC

.MO

D—

—1

32

9.7

61

.62

7.3

63

.01

32

9.7

61

.62

7.3

63

.01

32

9.7

61

.62

7.3

63

.0(3

-co

mp

.)2

34

4.76

1.3

42

.16

5.2

23

44

.76

1.3

42

.16

5.2

23

44

.76

1.3

42

.16

5.2

33

61

.16

4.0

30

.66

2.2

33

61

.16

4.0

30

.66

2.2

33

61

.16

4.0

30

.66

2.2

16

3V

SC

.UR

34

9.56

0.5

13

31

.26

3.2

9.6

60

.11

33

8.6

60

.91

9.6

64

.51

33

6.1

64

.41

6.2

69

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33

1.2

63

.29

.66

0.1

23

53

.56

3.4

90

.46

0.1

23

54

.86

0.1

80

.46

4.5

23

54

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0.7

83

.86

9.2

23

53

.56

3.4

90

.46

0.1

Info

rmat

ion

onde

natu

red

prot

eins

appe

ars

inita

lics.

*Flu

ores

cenc

ew

asex

cite

dat

280

nm.

1726 Biophysical Journal Volume 81 September 2001 1710–1734

Page 18: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

the calculation data for native (N) and denatured (D)proteins. The histograms accounting only for the differ-ences in contributions of individual components in wholespectra are presented in Fig. 4 (s(i) 5 1.0 nm; S(i) 5var). The resulting reduction in contributions of “small”components (S(i) , 0.01) in the histogram essentiallyimproved the resolution of individual peaks in it. The“small” components may not only have worse determinedlm values, but also may be erroneous in principle in thecases of bad quality of a spectrum under decomposition.The fact that the improved resolution of peaks in suchhistograms at;326, 331, 345, and 350 nm allowed us toassume that they reflect the existence of discrete classesof tryptophan emitters in native proteins. The reductionof contributions of minor components essentially de-creased the relative amplitude of a shorter-wavelengthwing in the denatured-proteins’ histogram. The remain-ing shorter-wavelength peaks (instead of a “continuum”)roughly correspond to the peaks in the native-proteins’histogram. However, the 350-nm peak and the shoulder at340 –347 nm became much better-expressed.

To demonstrate that the resolution of peaks obtained inprevious histograms (and, thus, the observed spectraldiscrete classes) are beyond the error of the estimation ofthe maximum position of spectral components, we con-structed the histograms (Fig. 5) accounting for the pre-cision of lm(i) estimation (s(i) 5 var) and the relativecontributions of components (S(i) 5 var). These histo-grams demonstrate the presence of discreteness of emis-

sion of tryptophan residues in proteins in the same man-ner as Figs. 3 and 4. There are seen the global minimumat ;337 nm and four maxima at;326, 333, 344, and 350nm for native proteins. The PHREQ algorithm well-resolves only two classes, because it was applied only tothe limited number of protein spectra, possessing one ortwo components. For denatured proteins, accounting fors(i) made peaks even more expressive. The main maximafor denatured proteins are at;346 and 350 nm; however,the peak at;334 nm also exists.

DISCUSSION

In 1973–1977 Burstein and co-workers postulated the hy-pothesis of the existence of discrete classes of tryptophanresidues in proteins (Burstein et al., 1973; Burstein, 1977a,1983). Later, algorithms of stable and reliable decomposi-tion of composite tryptophan fluorescence spectra into log-normal components were developed (Abornev and Burstein,1992; Burstein and Emelyanenko, 1996; Burstein et al.,2001) that allowed us to verify and revise the hypothesis.Here, we used mathematically different algorithms of de-composition of emission spectra to examine the stability ofsolutions. The fitting algorithm (SIMS) and the analyticalones (PHREQ) gave, in general, very similar results: ob-tained maximum positionslm(i) differed within an error of62.8 nm, and contributionsS(i) of components differedwithin the range of68.6%.

FIGURE 1 Canonical distribution of occurrence of maximum positions of total, non-decomposed emission spectra for native (A) and denatured (B)proteins with 2-nm step. Solid lines represent the fitting by Gauss function.

Discrete States of Tryptophan Residues 1727

Biophysical Journal 81(3) 1710–1734

Page 19: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

First of all, we showed that the distribution of maximumpositions of total, nondecomposed spectra (“lm total” inTable 2) for native proteins is rather well-fitted by a Gaussequation with the maximum at;337 nm and dispersion of;12 nm (Fig. 1A), which demonstrated the randomnessand, thus, representativeness of the set of the proteins understudy. Then, we analyzed the distributions of the maximumposition of log-normal components taking into consider-

ation the experimental deviations of maximum position andthe contributions of components to spectra of native anddenatured proteins (Figs. 2–5). All obtained data confirmedthe existence of several statistically discrete classes of emit-ting tryptophan fluorophores in proteins. The discreteclasses observed here are in approximate agreement withthose proposed in 1973–1977 in the model of discrete states.A very important point is that there was no a priori assump-

FIGURE 2 Canonical distribution of occurrence of maximum positions of log-normal components obtained by applying SIMS (A), PHREQ (B)algorithms, and different schemes of averaging of the results: “Average” (C) and “Mean Best Fit” (D) for native proteins with 2-nm step.

1728 Reshetnyak and Burstein

Biophysical Journal 81(3) 1710–1734

Page 20: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

tion about the discreteness in any algorithm used for compo-nent analysis of fluorescence spectra (Burstein et al., 2001).

It is evident that to find the causes for so widely differingand discrete Stokes shifts in the emission spectra of trypto-phan fluorophores in proteins one has to look in a variety ofcombinations of interactions of individual fluorophores withtheir environment, occurring mainly during the lifetime of

the fluorescent excited state. The physical causes for thedifferences among the spectroscopic classes can be ana-lyzed in terms of various combinations of Bakhsiev’s theoryof specific and universal interactions of the excited fluoro-phores with their environment (Bakhshiev, 1972). Bothkinds of interactions are induced by essential redistributionof electronic density on the atoms and chemical bonds of

FIGURE 3 Distribution of occurrence of maximum positions of log-normal components obtained by applying SIMS (A), PHREQ (B) algorithms, anddifferent schemes of averaging of the results: “Average” (C) and “Mean Best Fit” (D) with 1-nm step. Each curve is a sum of Gauss function (seeexplanation in the text) for each spectral component of native (N) and denatured (D) proteins withs(i) 5 1.0 andS(i) 5 1.0.

Discrete States of Tryptophan Residues 1729

Biophysical Journal 81(3) 1710–1734

Page 21: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

fluorophore after its electronic excitation. The specific in-teractions include changes in the nearest range interactionswith neighboring groups and/or solvent molecules, i.e., re-solvation, hydrogen bond formation, or other noncovalentcomplexing (Bakhshiev, 1972; Mataga et al., 1955). Theuniversal interactions occur due to the dipole relaxation of

surrounding dielectric continuum in response to the changesin direction and magnitude of fluorophore dipole moment atthe excitation (Bakhshiev, 1972; Bilot and Kawski, 1962;Lippert, 1957; Liptay, 1965;. Mataga et al., 1956).

The formation of stoichiometric complexes (most proba-bly hydrogen-bonded ones) of excited indole fluorophore

FIGURE 4 Distribution of occurrence of maximum positions of log-normal components obtained by applying SIMS (A), PHREQ (B) algorithms, anddifferent schemes of averaging of the results: “Average” (C) and “Mean Best Fit” (D) with 1-nm step. Each curve is a sum of Gauss function (seeexplanation in the text) for each spectral component of native (N) and denatured (D) proteins withs(i) 5 1.0 andS(i) equal the relative contribution ofthe component in the total protein emission (see Table 2).

1730 Reshetnyak and Burstein

Biophysical Journal 81(3) 1710–1734

Page 22: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

with alcohols was demonstrated experimentally (Walker etal., 1967; Lumry and Hershberger, 1978; Hershberger et al.,1981). Such complexes with alcohols in apolar cyclohep-tane had discrete positions of fluorescence spectral maximadependent on the stoichiometric ratios of alcohol/fluoro-phore: the maximum was at 316 nm for the 1:1 ratio and at

;330 nm at the 2:1 ratio compared with the central peak at307 nm in the structured spectrum obtained in the absenceof any polar co-solvents. The authors interpreted these ex-cited-state complexes (exciplexes) as a result of H-p-bond-ing of alcohol hydroxyls with indolic N- and Cg-atoms asthose possessing maximal electronic density in the excited

FIGURE 5 Distribution of occurrence of maximum positions of log-normal components obtained by applying SIMS (A), PHREQ (B) algorithms, anddifferent schemes of averaging of the results: “Average” (C) and “Mean Best Fit” (D) with 1-nm step. Each curve is a sum of Gauss function (seeexplanation in the text) for each spectral component of native (N) and denatured (D) proteins withs(i) equal to the standard (root-mean-square one) errorof the meanlm(i) value andS(i) equal to the relative contribution of the component in the total protein emission (see Table 2).

Discrete States of Tryptophan Residues 1731

Biophysical Journal 81(3) 1710–1734

Page 23: Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. II. The Statistical Proof of Discreteness of Tryptophan Classes in Proteins

state. Such a location of H-bonds may be revised in the lightof recent quantum-mechanical calculations of electronicdensity distribution in the excited indole and tryptophanfluorophores, which revealed that maximal densities arelocated at the Ce3, Cz2, and Cd2 atoms in the main fluo-rescent1La state of the indolic ring (Callis, 1997).

In proteins, the structured fluorescence bands coincidingwith those of nonexciplexed indole fluorophores occur inazurin (Burstein et al., 1977) and in bacteriorhodopsin pur-ple membranes suspended in 2 M CsCl (Permyakov andShnyrov, 1983). Such tryptophan residues were attributed toclass A in the hypothesis of discrete states. The structuredspectra similar to those of the exciplexes 1:1 occurring inseveral single-tryptophan-containing proteins were assignedto class S (Burstein, 1977a, b, 1983). Emission spectra ofclasses A and S do not undergo any spectral shift underfreezing the protein solutions down to2196°C, i.e., thedipole moments of these fluorophores practically do notchange at the excitation. In the histograms obtained in thiswork, classes A and S are reflected by peaks and/or shoul-ders at 305–308 and 316–318 nm, respectively (see Figs.3–5).

In the model experiments (Walker et al., 1967; Lumryand Hershberger, 1978; Hershberger et al., 1981), the max-imum position of the exciplex 2:1 spectrum was not con-stant under increasing alcohol concentration, which re-flected the rise of Stokes shift induced by the solvent dipolerelaxation in response to the change of fluorophore dipolemoment in the excited state of the exciplex. Therefore, theflorescence of protein tryptophan residues possessing thespectra with maxima at;330 nm and longer were assignedto the emission of exciplexes with stoichiometry not,2:1in the model of discrete classes (Burstein, 1977a, b, 1983).The model provided three discrete classes (I–III) for suchexciplexes with stoichiometry$2:1, differed in their acces-sibility to extrinsic quenchers (vanishing accessibility forclass I,lm at ;330 nm, and the accessibility of.30% ofthat of free tryptophan for classes II and III). The differencebetween two accessible classes was assumedly interpretedas a result of different dipole relaxation rates in their envi-ronments.

The histograms (Figs. 3–5) contain peaks reflecting allthree discrete classes. However, the class I band in thehistograms splits up into two peaks, at;325 and 332 nm.Almost all histograms contain the global minimum at 336–337 nm and two evident maxima at 344–345 and 349–351nm, which correspond to the emission of exposed trypto-phan residues of classes II and III, respectively. In themodel of discrete classes, it was suggested that exposedtryptophan residues in native proteins should emit at 340–342 nm, and emission at 350–353 nm is due to tryptophanresidues of denatured proteins (Burstein et al., 1973). How-ever, the extended set of proteins investigated here indicatesthat tryptophans in native proteins also can have spectrawith maxima at;350 nm.

Tryptophan residues of denatured proteins (italicized inTables 1 and 2) have the main peak positions at 340–345and 351 nm. The longest-wavelength peak corresponds tothe completely unfolded proteins in urea or guanidiniumchloride solutions. However, the peak at 340–345 nm be-longs mainly to the fluorophores in proteins denatured byextreme pH values (e.g., pepsin at pH 6–9; see Table 2) orhigh dioxane concentrations (chymotrypsin, chymotrypsin-ogen, and trypsin; see Table 2). It is possible that suchmaximum positions of fluorescence spectra are characteris-tic of tryptophan residues in such intermediate unfoldingstates of proteins as molten globule (Ptitsyn, 1995). Theshorter-wavelength peaks (lm , 337 nm) in histograms fordenatured proteins belong, most probably, to the fluoro-phores in molecules retained native or partly unfolded indenaturing conditions.

The model of discrete states reflects the existence inproteins of the five most manifested classes of tryptophanresidues. The discreteness of fluorescence parameters has tohave a probabilistic nature. The histograms obtained in thiswork reflected all these classes and can be regarded as astatistical confirmation of the hypothesis of discrete classesof tryptophan residues in proteins. It could be assumed thatsuch a situation might be realized because tryptophan resi-dues are located in a few kinds of physically preferredenvironments in protein structures that provide realizationof different combinations of specific and universal interac-tions of excited fluorophore with its environment.

There were a lot of papers published during the lastseveral last years where it was demonstrated that tryptophanresidues could have various fluorescence parameters de-pending on their environment in proteins (Callis, 1997;Callis and Burgess, 1997; Reshetnyak and Burstein, 1997a,b; Chen and Barkley, 1998; Kuznetsova and Turoverov,1998; Meagher et al., 1998). Because highly resolved x-rayand NMR structures of many proteins are now available, wedeveloped a system of describing physical and structuralcharacteristics of the microenvironment of tryptophan resi-dues and examined the existence of discreteness of struc-tural parameters of the microenvironment of tryptophanresidues in proteins. Moreover, that work allowed us toelucidate the main physical and structural factors determin-ing the class of an individual tryptophan residue. The nextpaper of this series contains the results of that study.

The authors are thankful to all colleagues from the Laboratory of ProteinFunctional Biophysics of the Institute of Theoretical and Experimental Bio-physics (Pushchino) for everyday help and discussions. We thank Drs. K. K.Turoverov and R. V. Polozov for fruitful discussions. We are especiallygrateful to Drs. V. M. Grishchenko, L. P. Kalinichenko, and T. G. Orlova forthe help in experimental work and to Drs. D. B. Veprintsev and D. S. Rykunovfor valuable consultations and maintaining the function of the computers andnets. We are much obliged to all colleagues who placed protein preparationsand/or fluorescence spectra at our disposal.

This work was supported in part by Grants 95-04-12935, 97-04-49449, and00-04-48127 from the Russia Foundation of Basic Research.

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