A spectroscopic probe of stacking interactions between nucleic acid bases and tryptophan residues of...

volume 7 Number 71070 Nucleic Ac ids Research

A spectroscopic probe of stacking interactions between nucleic acid bases and tryptophanresidues of proteins

Claude Helene, Jean-Jacques Toulme and Trung Le Doan

** Centre de Biophysique Moleculaire, CNRS, 45045 Orleans Cedex and Laboratoire de Biophysique,Museum National d'Histoire Naturelle, 61, rue Buffon, 75005 Paris, France

Received 25 September 1979

ABSTRACT

The external heavy atom effect of mercury on the spectros-copic properties of the indole ring has been used to investigate stackinginteractions of tryptophan with mercurinucleotides in mixed aggregates for-med in frozen aqueous solutions as well as in oligopeptide-polynucleotidecomplexes. This effect is characterized at 77 K by a quenching of the tryp-tophan fluorescence, an enhancement of the phosphorescence emission and adrastic shortening of the phosphorescence lifetime. These phenomena resultfrom an enhanced spin-orbit coupling due to a close contact between the mer-cury atom and the indole ring. Dissociation of the complexes leads to a r e -covery of the spectroscopic properties of the free tryptophan ring. The pos-sible use of this spin-orbit probe to provide evidence for stacking interac-tions in protein-nucleic acid complexes is discussed.

INTRODUCTION

Stacking interactions involving aromatic amino acids and nu-

cleic acid bases might play an important role in the formation of protein-

nucleic acid complexes (1 ,2) . It has been suggested that such interactions

might be involved in the selective recognition of single stranded structures

of nucleic acids by proteins (3, 4). However it is always difficult to provide

direct evidence for the involvement of stacking interactions in a biologically

important complex. Nuclear magnetic resonance ia probably the most ap-

propriate method since stacking induces upfield shifts of the proton resonan-

ces of the stacked aromatic rings (5). However this method can be easi ly

used only with simple systems because usually the presence of several aro-

matic amino acids in a protein leads to a superimposition of the aromatic

proton resonances which preventeany unambiguous analysis of changes in che-

mical shifts. The replacement of tyrosines by fluorotyrosines or by s e -

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Nucleic Acids Research

lectively deuterated tyrosines has made it possible to study stacking interac-19 1 .

tions by F and H magnetic resonance in the complexes formed by oligo-

nucleotides with gene 5 protein from phage fd (6). However these methods

require the preparation of labeled proteins and the number of cases where

they can be applied is therefore limited.

Fluorescence spectroscopy has been widely used to study

protein-nucleic acid complexes (7). Stacking interactions are characterized

by a quenching of tryptophyl, tyrosyl Or phenylalanyl fluorescence (4, 8).

However there might be other mechanisms by which the fluorescence of aro-

matic residues could be quenched. A change of the protein conformation

might bring the aromatic residue in the vicinity of a quenching group inside

the protein structure. Also energy transfer from tyrosine and phenylalanine

to nucleic acid bases might contribute to fluorescence quenching in the ab-

sence of stacking interactions (4).

Recently a photochemical method has been developed which

makes use of the photosensitizing properties of tryptophyl residues. It has

been shown that tryptophan is able to photosensitize the splitting of thymine

dimers in oligopeptide-DNA or protein-DNA complexes (9). This reaction

requires a close proximity of tryptophan and dimers. However the prerequi-

site is that thymine dimers are first made by UV irradiation of DNA. This

might alter the recognition of DNA by the investigated protein.

Photochemical cross-linking of tryptophan to nucleic acid ba-

ses in a stacked complex could possibly help identify a stacking interaction.

No such reaction has been described yet with the possible exception of the

photochemical reaction of tryptophan with 5-bromouracil (10). This possi-

bility is presently under investigation.

We have tried to use a direct spectroscopic method to provide

evidence for stacking interactions. This method is based upon the external

heavy atom effect on spin-orbit coupling in aromatic molecules (11) . It is

known that spin-orbit coupling can be strongly enhanced by Van der Waals

contact of the aromatic molecule with a perturbing heavy atom. Such an ef-

fect has already been used to investigate intercalation of acridine dyes in

polynucleotides containing 5-bromouracil (12) and more recently to probe

the presence of a tryptophan residue in the active site of a lectin by using a

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ligand containing a mercury atom (13). The heavy atom effect is characte-

rized by a quenching of the fluorescence of the aromatic molecule, an en-

hancement of the phosphorescence emission and a shortening of the phospho-

rescence lifetime. It has been shown that mercurinucleotides can be incor-

porated in polynucleotide chains (14). We have therefore investigated the

possibility of using the heavy atom effect of mercury to provide evidence

for stacking interactions of mercurinucleotides with tryptophan. The results

presented below show that such a heavy atom effect can be observed in two

systems : i) in mixed aggregates formed when aqueous mixtures of trypto-

phan (or other indole derivatives) and 5-mercuri dUMP are frozen down to

77 K ; ii) in the complexes formed by the oligopeptide Lys-Trp-Lys with

mercurated poly(U).

MATERIALS AND METHODS

Tryptophan, N-acetyltryptophan amide, indole, were obtai-

ned from Sigma. N-methyl indole distilled under vacuum was a gift from

Dr. J. P. Privat. N(l)-methyltryptophan was synthesized by Dr. M. Bazin.

5-mercuri deoxyuridine, 5'-monophosphate was obtained

from P. L. Biochemicals as the triethyl ammonium salt of the mercuri car-

bonate derivative of the nucleotide. Two samples of mercurated poly(U)

were used : one was a gift from Drs. M. N. Thang and J. L. Drocourt, the

other was obtained from P. L. Biochemicals. Both gave identical results.

The percentage of uracil bases substituted by mercury at carbon 5 was hi-

gher than 70 %.

Luminescence measurements at 77 K were carried out with

a Jobin-Yvon Bearn spectrofluorimeter modified to correct for lamp fluc-

tuations (8). The solutions were contained in a quartz tube of 2 mm i. d. im-

mersed in a quartz dewar containing liquid nitrogen. Phosphorescence was

separated from fluorescence by a rotating can phosphoroscope which was

used as a shutter for fast decay time measurements.

RESULTS

1. Frozen aqueous solutions of 5-HgdUMP and indole derivatives

Freezing an aqueous solution induces the formation of aggre-

gates of the solute molecules (15). When an aqueous mixture of two aroma -

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tic molecules is frozen down to 77 K mixed aggregates are usually formed.

This property has been previously used to study stacking interactions bet-

ween aromatic amino acids and nucleic acid bases (16).

When an aqueous equimolecular mixture of N-Acetyl trypto-

phan amide (NATA) and 5 HgdUMP was frozen down to 77 K, a complete

quenching of indole fluorescence was observed together with the appearance

of a strong phosphorescence emission of short lifetime (figure 1, table 1).

The phosphorescence spectrum had a vibronic structure which resembled

that of NATA dispersed in a frozen glass of water and propyleneglycol

(lv/lv) (WPG) although it was red-shifted by about 16 nm. It should be no-

ted that NATA aggregates (in the absence of 5 HgdUMP) emit a strong fluo-

rescence but only a very weak phosphorescence.

In order to determine the origin of these spectroscopic effects

several derivatives of indole and uridine were investigated. Uridine itself

quenched the fluorescence of NATA and tryptophan but no phosphorescence

3H«dUHP

400 500 X (nm)

Figure 1 : Total luminescence spectra in frozen aqueous solutions at 77 Kof N-acetyltryptophanamide (NATA) ( . . . ) , 5 HgdUMP ( ) and their equi-molar mixture ( ). The concentrations were 3. lxlO'^M for each com-pound. Note that the spectra of the individual molecules are multiplied by 3.The spectrum -o-o- represents the phosphorescence emission of NATA in awater-propylene glycol (lv/lv) glass.

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Table I : Phosphorescence lifetimes at 77 K in frozen aqueous solutions (W)and in frozen equivolume mixture of water and propylene glycol (WPG). Thephosphorescence decays were usually not strictly exponential except for5HgdUMP in water and NATA in WPG. The lifetimes given here correspondto the initial parts of the decay curves.

5 HgdUMP Tryptophan NATA NATA NATA+ 5 HgdUMP + 5 HgdUMP + 5 HgdUMP

(W) (W) (W) (WPG) (WPG)

2. 7 m s 2. 9 m s 3 . 7 m s 6. 2 s 6. 1 s

Indole + N-methyl indole N(l) methyl tryptophan5 HgdUMP + 5 HgdUMP + 5 HgdUMP

(W) (W) (W)2.4 ms 2 .7 m s 2.9 ms

enhancement was observed as already reported (16). A similar result was

obtained with 5-bromodeoxyuridine. Fluorescence quenching was previously

ascribed to the formation of electron donor-acceptor complexes in aggrega-

tes (16). The following indole derivatives were also investigated : indole,

N-methylindole, tryptophan,N(l)-methyltryptophan. Equimolar mixtures of

these compounds with 5-HgdUMP all gave rise to the spectroscopic effects

described above for NATA. They were characterized by a quenching of the

indole fluorescence, the appearance of an intense phosphorescence with a

vibronic structure characteristic of indole derivatives and a very short

phosphorescence lifetime of the order of 3 ms (table 1). In the absence of

5-HgdUMP the phosphorescence emitted by indole derivatives in frozen

aqueous solutions was too weak to permit a precise measurement of its de-

cay. However this phosphorescence was easily measured in a frozen glass,

e.g., a mixture of water and propylene glycol (lv/lv). Its decay was expo-

nential with a lifetime of 6-7 s for all indole derivatives (17). The interac-

tion with 5 HgdUMP in aggregates therefore reduced the lifetime by a factor

of about 2, 000.

It is known that addition of organic solvents to aqueous solu-

tions before freezing prevents the formation of aggregates (15). Addition of

an equivolume of propylene glycol to an equimolar mixture of NATA and

5 HgdUMP suppressed the spectroscopic effects described above. The fluo-

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rescence of NATA was not quenched, the normal phosphorescence spectrum

and lifetime were restored. This experiment demonstrates that aggregate

formation is required to observe the heavy atom effect of 5 HgdUMP on

NATA.

2. Complexes of Lys-Trp-Lye with mercurated poly(U)

We have previously shown that the tryptophyl ring of the tri-

peptide Lys-Trp-Lys forms stacked complexes with nucleic acid bases when

it binds to polynucleotides (5,7). We have therefore investigated the comple-

xes formed by Lys-Trp-Lys with mercurated poly(U). As shown in figure 2,

the formation of this complex is characterized by a quenching of Trp fluo-

rescence, an enhancement of the phosphorescence quantum yield together

with a drastic shortening of the phosphorescence lifetime (table 2). It should

be noted that the phosphorescence spectrum is red-shifted by 8 nm. This

red-shift is smaller than that observed in aggregates (see above).

Complex formation between Lys-Trp-Lys and polynucleotides

involves electrostatic interactions between amino groups (N-terminal and

20

r,

10 -

Ly»-Tpp4_y»

/ \

. - - - ^ ••—#'

r ~ i

r\ Ly»-Tr-|>4_ym <• Poly HgU

j\\ \Poly HgU \

1 1 I

300 400 500 X ( nm)

Figure 2 : Total luminescence spectra at 77 K in a pH 6 buffer (B) contai-ning 1 mM sodium cacodylate and 1 mM sodium chloride of 8x10"^ M Lys-Trp-Lys ( . . . ) , 3.3xlO-4 M poly HgU ( ) and the mixture of 8x10-5 MLys-Trp-Lys with 3.3xlO"4 M poly HgU ( ).

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- 4Table 2 : Phosphorescence lifetimes of poly(5 HgU) (1. 7x10~ M). Lys -Trp-Lys (3.4xlO"5 M) and their mixture in a buffer (B) containing 1 mMsodium cacodylate, 1 mM sodium chloride, pH 6 or in an equivolume mix-ture of buffer and propylene glycol (WPG)

poly(5 HgU)

(B)

1. 8 ms

Lys-Trp-Lys+ poly(5HgU)

(B)

5. 9 ms (82%)+ 5. 1 e (18%)

Lys-Trp-Lys

(WPG)

6.2 s

• pol^u]

(WPG)

5. 6 ms (85%)+ 6.0s (15%)

Lys-Trp-Lys+ poly(5 HgU)

(WPG)

6. 0 s

lysyl side chains) and phosphate groups together with a stacking of Trp with

bases (5, 8). These complexes are dissociated when the ionic strength in-

creases. Figure 3 shows the results of an experiment conducted in the sol-

vent WPG which forms a transparent glass at 77 K and does not prevent

complex formation between Lys-Trp-Lys and polynucleotides. Increasing

the ionic strength from 1 mM to 400 mM leads to a reversal of the effects

described above : the original fluorescence of the tryptophyl residue is re-

Poly HgU • Lys-Trp.Lys

. „ . Lys-Trp-Ly»

_ Poly HgU* LyiTrp Lys• O.«MNaCI

350 450 550

Figure 3 : Total luminescence spectra in an equivolume mixture of propy-lene glycol and buffer B (see legend of figure 2) at 77 K of 4xlO"5 M Lys-Trp-Lys ( . . . ) , and the mixture of 4xlO"5 M Lys-Trp-Lys with 1. 6x10-4 Mpoly HgU in the absence ( ) and in the presence ( ) of 0.4 M NaCl. Itshould be noted that the phosphorescence of HgU is superimposed on that ofLys-Trp-Lys when the complex with poly(HgU) is dissociated in the presen-ce of 0.4 M NaCl (---).

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covered while the phosphorescence quantum yield is decreased and the phos-

phorescence lifetime returns to that of the free peptide (table 2).

It should be noted that the phosphorescence decay of Lys-

Trp-Lys complexes with poly 5 HgU shows a long-lived component (w 5-6 s)

superimposed on the main short-lived decay. The contribution of this long-

lived component (w 15 %) is too high to be explained only by the presence of

unbound or externally bound peptides (8). Since the poly U samples used in

our experiments are not fully mercurated it is likely that some tryptophyl

residues are stacked with non-mercurated uracils and that the triplet state

of these residues is efficiently populated via triplet energy transfer from

neighboring bases. However due to the absence of a mercury atom in their

immediate vicinity the phosphorescence of these tryptophyl residues is ex-

pected to have *he same lifetime as unperturbed tryptophan (table 2). Such

an efficient triplet energy transfer from adenine to tryptophan was already

observed in the complexes formed by Lys-Trp-Lys with poly(A) (20).

DISCUSSION AND CONCLUSION

The results obtained both in mixed aggregates and in Lys-Trp-

Lys complexes with mercurated poly(U) clearly demonstrate the influence of

the mercury atom on the spectroscopic properties of the indole ring. The

enhancement of spin orbit coupling due to the heavy atom effect is characte-

rized by a complete quenching of the indole fluorescence, a strong increase

of the phosphorescence emission and a drastic shortening of the phospho-

rescence lifetime.

The fact that the same phenomenon is observed in mixed ag-

gregates when the N(l) atom of the indole ring is methylated or when the

amino and carboxyl groups of the amino acid function of tryptophan are sub-

stituted, indicates that the spectroscopic changes are due to a direct effect

of the mercuri nucleotide on the indole ring, and do not involve binding of

mercury to the amino group as reported for complexes of tryptophan or

tryptamine with CH HgOH (18). These spectroscopic changes are charac-

teristic of the heavy atom effect of mercury on spin-orbit coupling in the

excited indole derivative. They require a Van der Waals contact between

the mercury atom and the indole ring (11). Since it is known that tryptophan

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forms stacked complexes in mixed aggregates with nucleosides in frozen

aqueous solutions it is very likely that the contact between the mercury atom

and the indole ring results from such a stacking interaction.

In the complexes formed by Lys-Trp-Lys with mercurated

poly(U) at pH 6 the amino groups of lysyl residues are positively charged

and interact strongly with the negatively charged phosphates (rather than

form complexes with the mercury atom) (8). The behavior of these comple-

xes is very similar to that observed with other polynucleotides, including

their dissociation at high ionic strength (7). Since proton magnetic resonan-

ce experiments have clearly shown that the tryptophyl ring of the tripeptide

is stacked with nucleic acid bases (5), it is very likely that the same interac-

tions are involved in the complexes described here. The heavy atom effect

on the spectroscopic properties of the tryptophyl residue is thus due to the

close contact between the mercury atom and the indole ring brought about by

the stacking of tryptophan and uracil. The red shift of the phosphorescence

spectrum as compared to free tryptophan is very likely due to this stacking

interaction as already observed in Lys-Trp-Lys complexes with poly(A) (20]l

as well as to an effect of mercury itself (18).

This heavy atom effect should be easily detected in protein-

nucleic acid complexes if stacking interactions are involved. Of course the

presence of mercuri nucleotides in the nucleic acid is a prerequesite. It

will be necessary to prevent reaction of sulfhydryl groups of the protein (if

present) with the mercurinucleotide. This can be achieved either by reaction

of mercaptans with the mercuri-substituted nucleic acid before complex for-

mation or by protection of the protein SH groups if this does not prevent bin-

ding to the nucleic acid.

Acknowledgements

We widh to thank Drs. M. N. Thang and J. L. Drocourt for a

gift of mercurated poly U and Dr. M. B&zin for a gift of N(l)-methyltrypto-

phan. This work was supported in part by a grant (MBM 77-7-1741) of the

Delegation Ge'ne'rale a la Recherche Scientifique et Technique (DGRST).

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