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The Protein Journal ISSN 1572-3887Volume 34Number 1 Protein J (2015) 34:60-67DOI 10.1007/s10930-014-9597-3

Acid Stability of the Kinetically StableAlkaline Serine Protease PossessingPolyproline II Fold

Sonali Rohamare, Vaishali Javdekar,Sayli Dalal, Pavan Kumar Nareddy,Musti J. Swamy & Sushama M. Gaikwad

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Acid Stability of the Kinetically Stable Alkaline Serine ProteasePossessing Polyproline II Fold

Sonali Rohamare • Vaishali Javdekar •

Sayli Dalal • Pavan Kumar Nareddy •

Musti J. Swamy • Sushama M. Gaikwad

Published online: 10 January 2015

� Springer Science+Business Media New York 2015

Abstract The kinetically stable alkaline serine protease

from Nocardiopsis sp.; NprotI, possessing polyproline II

fold (PPII) was characterized for its pH stability using

proteolytic assay, fluorescence and Circular Dichroism

(CD) spectroscopy, and Differential Scanning Calorimetry

(DSC). NprotI was found to be functionally stable when

incubated at pH 1.0, even after 24 h, while after incubation

at pH 10.0, drastic loss in the activity was observed. The

enzyme showed enhanced activity after incubation at pH

1.0 and 3.0, at higher temperature (50–60 �C). NprotI

maintained the overall PPII fold in broad pH range as seen

using far UV CD spectroscopy. The PPII fold of NprotI

incubated at pH 1.0 remained fairly intact up to 70 �C.

Based on the isodichroic point and Tm values revealed by

secondary structural transitions, different modes of thermal

denaturation at pH 1.0, 5.0 and 10.0 were observed. DSC

studies of NprotI incubated at acidic pH (pH 1.0–5.0)

showed Tm values in the range of 74–76 �C while signifi-

cant decrease in Tm (63.8 �C) was observed at pH 10.0.

NprotI could be chemically denatured at pH 5.0 (stability

pH) only with guanidine thiocynate. NprotI can be classi-

fied as type III protein among the three acid denatured

states. Acid tolerant and thermostable NprotI can serve as a

potential candidate for biotechnological applications.

Keywords Acid stability � Kinetic stability � Polyproline

fold � Protease � Thermal unfolding � DSC

Abbreviations

PPII Polyproline II

CD Circular Dichroism

DSC Differential Scanning Calorimetry

NprotI Nocardiopsis protease I

GuSCN Guanidine thiocyanate

GdnHCl Guanidine hydrochloride

MRE Mean residual ellipticity

ANS 1-Anilino 8-napthalene sulfonic acid

Napase Nocardiopsis alba protease

1 Introduction

Many of the extracellular proteases of microbial and plant

origin have undergone modification during evolution e.g.

alpha lytic protease [1], milin [2] to remain stable and

functional under various harsh environmental conditions

such as high temperature, low pH (acidic medium), or

presence of other proteases. These proteases are called as

kinetically stable proteases. Studying the mechanisms or

structural factors underlying the stability of these proteins

is of great importance to understand protein folding and to

develop protein engineering strategies. In a previous study

[3], we reported the presence of polyproline fold (PPII) in a

serine protease (NprotI) from Nocardiopsis sp. NCIM

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10930-014-9597-3) contains supplementarymaterial, which is available to authorized users.

S. Rohamare � S. Dalal � S. M. Gaikwad (&)

Division of Biochemical Sciences, National Chemical

Laboratory, Dr. HomoBhabha Road, Pune 411008, India

e-mail: sm.gaikwad@ncl.res.in

V. Javdekar

Department of Biotechnology, Abasaheb Garware College,

Pune, India

P. K. Nareddy � M. J. Swamy

School of Chemistry, University of Hyderabad,

Hyderabad 500046, India

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Protein J (2015) 34:60–67

DOI 10.1007/s10930-014-9597-3

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5124. The PPII fold was implicated in assigning resistance

to the protein against GdnHCl denaturation, proteolytic

digestion and solvent denaturation.

In the present studies, unusual stability of NprotI in the

extreme acidic pH range (pH 1.0–3.0) at 50�–60 �C has

been observed using proteolytic assay and biophysical

techniques. The low pH conditions have made acidophilic

proteins to function solely in acidic environment with little

survival or loss of functionality in neutral and alkaline

conditions. The optimum conditions for Nocardiopsis sp.

NCIM 5124, for production of NprotI require pH 10.0.

Also, NprotI shows maximum activity at pH 10.0. However,

the enzyme is most stable at pH 5.0. The same trend

(optimum activity in alkaline range, stability in acidic

range) was also observed in a serine protease from N. alba

[4]. Presence of certain nutrients causes the Nocardiopsis

sp. to greatly acidify their environment. Thus, exposure to

low pH, even temporararily, might necessitate development

of secreted proteins with stability and activity at low pH.

pH of the medium is a major factor influencing protein

stability as it affects ionic interactions. Also, thermal

denaturation of proteins can be affected by changes in pH,

by changing the conformational energy differences

between the native and denatured states [5].

The effect of pH on the stability of PPII fold has not

been studied extensively as yet. In the present report, the

conformational (w.r.t. PPII fold) and functional stability of

NprotI was studied over a broad pH range. The overall

structure of NprotI was stable in the pH range of 1–12,

while the enzyme showed functional stability after longer

time incubation only in acidic pH range (when assayed at

pH 10.0). Also, a pH dependence of thermal unfolding was

studied using Circular Dichroism (CD) and Differential

Scanning Calorimetry (DSC).

2 Materials and Methods

2.1 Materials

Guanidine hydrochloride (GdnHCl) and guanidine thiocy-

ante (GuSCN) (Molecular Biology grade) were obtained

from Sigma Aldrich Ltd., USA. All other reagents and buffer

compounds were of analytical grade. MilliQ water was used

to prepare the solutions for spectroscopic measurements.

2.2 Production and Purification of NprotI

The production and purification of NprotI from Nocardi-

opsis sp. NCIM 5124 was performed by the protocol as

described earlier [6]. The purified enzyme exhibits maxi-

mum stability at pH 5.0, at 2–8 �C, hence was stored at

pH 5.0.

2.3 Enzyme Assay

Activity of the protease was determined by incubating 3 lg

of the enzyme in 300 ll of 1 % casein (substrate) at pH

10.0 at 60 �C for 20 min as described by Dixit et al. [6].

The amount of enzyme which releases 1 lmol of tyrosine

per minute in the assay conditions was considered as one

unit of protease activity.

2.4 Fluorescence Measurements

Perkin Elmer LS 50B luminescence spectrometer was used to

record steady-state fluorescence at 28 �C. The 30 lg/ml

protein solution in 20 mM acetate buffer pH 5.0 was excited at

295 nm. Emission was recorded in the range of 310–400 nm.

Scan rate of 100 nm/min was used to record the spectra. Slit

widths for excitation and emission monochromators were set

at 7 nm each. The signal produced by buffer solution was

subtracted, to eliminate background emission. All the exper-

iments were performed at least three times.

2.5 Circular Dichroism (CD) Measurements

A J-815 Spectropolarimeter with a PTC343 Peltier unit

(Jasco, Tokyo, Japan) was used to record CD spectra at

25 �C in a quartz cuvette. Each CD spectrum was accu-

mulated from five scans at 100 nm/min with a 1 nm slit

width and a time constant of 1 s for a nominal resolution of

0.5 nm. Protein solution of 250 lg/ml was used to collect

the far UV CD spectra for monitoring the secondary

structure. The wavelength range was of 200–250 nm and

path length of 0.1 cm. The enzyme (800 lg/ml) was used

to monitor tertiary structure with near UV CD spectra. The

wavelength range was 250–300 nm and path length was

1 cm. All spectra were corrected for buffer contributions

and observed values were converted to mean residue

ellipticity (MRE) in deg cm2 dmol-1 defined as

MRE ¼ Mhk=10dcr

where, hk is CD in millidegree, M is the molecular mass of

the protein, c is the protein concentration in mg/ml, d is the

path length in cm, and r is the average number of amino

acid residues in the protein.

2.6 ANS Binding Studies

The hydrophobic dye, 1-anilinonaphthalene 8-sulphonic

acid (ANS) binding study was used to analyze the inter-

mediate states of denatured and native NprotI incubated in

20 mM buffers of different pH (1–12). The final ANS

concentration used was 50 lM, excitation wavelength was

375 nm. Total fluorescence emission was monitored

between 400 and 550 nm.

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2.7 Effect of pH

The CD measurements were performed as described above.

For that samples of NprotI, (250 lg/ml) after incubation in

an appropriate buffer over the pH range of 1–12 for 6 h at

25 �C, were used. Prior to thermal denaturation experi-

ment, protein samples were incubated at respective pH for

4 h at 25 �C. The enzyme was then subjected to increasing

temperature with incubation time of 10 min at each tem-

perature before recording the CD scans. For activity stud-

ies, aliquots of enzyme were removed after suitable time

interval and assayed at optimum pH and temperature. The

following buffers (20 mM) were used for these as well as

for the DSC studies (Sect. 2.8): glycine–HCl for pH 1–3,

acetate for pH 4–5, phosphate for pH 6–7, Tris–HCl for pH

8–9 and glycine–NaOH for pH 10–12. pH of the reaction

remained stable even after 24 h. ANS binding studies were

performed as described in Sect. 2.6. All the experiments

were performed at least three times.

2.8 Differential Scanning Calorimetry

MicroCal VP-DSC differential scanning calorimeter

(MicroCal LLC, Northampton, MA, USA) equipped with

two fixed cells, a reference cell and a sample cell was used

for DSC measurements. A scan rate of 40 K/h was used for

DSC measurements at different pH. Desired pH was

achieved by dialyzing the samples extensively against

buffers of respective pH before recording the thermograms.

Degassing of buffer and protein solutions was done before

loading. Origin DSC software provided by the manufac-

turer was used for data analysis.

2.9 Effect of GuSCN

NprotI was incubated with various concentrations of

GuSCN (0-6 M) at pH 5.0, 25 �C and the enzyme assay

was performed after regular time interval.

3 Results

3.1 Stability of NprotI Over a Wide pH Range

3.1.1 pH Dependent Activity Profile of NprotI

Since the enzyme is best stored at pH 5.0, it was considered

to be the native condition of the enzyme. In all the studies,

activity of the enzyme was checked at pH 10.0 (optimum

pH) after incubation at different pH [3]. The pH activity

profile of NprotI is shown in Fig. 1a. Activity of the

enzyme incubated at pH 5.0 at zero time was taken as

100 %, as the enzyme is most stable at this pH for long

term storage [6]. Interestingly, the enzyme was found to be

more stable at extreme acidic pH, with increased activity

(*120 %) after incubation in the pH range of 1–3

(Fig. 1a). Incubation of NprotI alone at alkaline pH led to

drastic loss in the activity. This could be due to autoca-

talysis of the enzyme as it is optimally active at alkaline pH

and/or disruption of the active site geometry.

3.1.2 pH Dependent Structural Transitions in NprotI

3.1.2.1 CD Spectroscopy In previous work, it has been

shown that PPII fold is a unique structural feature of

NprotI, which is characterized by positive ellipticity at

230 nm and negative ellipticity below 200 nm in the far

UV CD spectrum of this enzyme [3]. In the present studies,

Fig. 1b shows that this structural element, especially

positive ellipticity around 230 nm remained stable over a

wide pH range while some alterations are seen in the

negative minima at different pH. At pH 1.0, the negative

ellipticity is reduced as compared to that observed at pH

5.0, although the minimum seen at 212 nm is sharp. While

in alkaline conditions, a trough rather than sharp minimum

was observed indicating some rearrangement in the struc-

ture (Fig. 1b), which led to comparatively unstable but

catalytically active structure.

3.1.2.2 ANS Binding Studies 1-Anilinonaphthalene 8-sul-

phonic acid (ANS) is a dye which shows increased fluores-

cence intensity when bound to hydrophobic regions of

proteins. This phenomenon has been widely used to detect

the molten globule state of different proteins. Fluorescence

intensity of ANS increased almost two fold in the presence of

NprotI with a concomitant blue shift in the kmax from 520 nm

to 485 nm at pH 5.0 (Fig. 1c) suggesting exposed hydro-

phobic amino acids even under native conditions which is

not commonly observed in proteins. The emission intensity

of ANS was found to increase further under acidic conditions

in the presence of NprotI, with the maximum increase being

observed at pH 1.0, indicating the possible presence of a

molten globule like structure, which could have led to more

functionally stable structure. The protein did not bind ANS at

pH 7.0 and above.

3.1.2.3 Near UV CD of NprotI The near UV CD spec-

trum of NprotI, at pH 5.0 shown in Fig. 1d indicated sig-

nificant ordered tertiary structure of the protein, which is

also retained at pH 1.0. The acid induced state appears to

retain a significant amount of native-like secondary and

tertiary structure, but noticeable amounts of exposed

hydrophobic side chains (Fig. 1c). This suggested the for-

mation of a compact ‘‘molten-globule’’- like intermediate

at acidic pH possessing constant secondary and compact

tertiary structure.

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3.1.2.4 Intrinsic Fluorescence Measurements The fluo-

rescence emission maximum wavelength, kmax (353 nm)

was not altered when NprotI was incubated in the pH range

of 1–12 indicating apparent conformational stability of the

protein in wide pH range (Fig S1). However, the emission

intensity was found to increase marginally at pH 1.0

whereas *20 % decrease was observed at pH 10.0 and

12.0. These observations suggested that at pH 1.0 the

microenvironment of the tryptophan residue is only mar-

ginally altered even after protonation of the surface amino

acid residues, whereas deprotonation of surface amino

acids of the protein result in quenching of the fluorescence

intensity at alkaline pH. There was no correlation between

the functional stability of the enzyme and fluorescence

profile.

3.2 Thermal Stability of NprotI At Different pH

3.2.1 Activity Studies

The unusual stability of NprotI at extreme acidic pH was

further investigated at higher temperatures. Interestingly,

NprotI showed 2.8–3.0 fold higher activity after incubation

at pH 1 and 3 as compared to that at pH 5.0, even after

incubation at 50 �C for 6 h (Fig. 2a) (all assayed at pH

10.0). The enzyme remained active at pH 1.0 for 24 h at

50 �C. For the sample incubated at pH 3.0, the

enhancement in activity was maintained for 10 h at 60 �C

(Fig. 2b). Rapid and total loss of activity of the enzyme

was observed after incubation at alkaline pH at 50 �C.

3.2.2 CD Spectroscopy of NprotI At Different pH

and Temperatures

Thermal denaturation of NprotI at pH 5.0, the native pH of

enzyme showed PPII fold melting above 65 �C and the

unfolding was irreversible [3]. In the present work, thermal

denaturation of NprotI at pH 1.0 and pH 10.0 was moni-

tored by recording far UV CD spectra at increasing tem-

perature. At pH 1.0, the PPII fold was fairly intact, up to

70 �C, the isodichroic point at 221 nm was observed till

70 �C (Fig. 3a). At pH 10.0, the protein started melting

above 60 �C (Fig. 3b) and the isodichroic point was not

observed, possibly due to the presence of more than two

conformational forms in the thermal denaturation process.

Around 65 �C the spectrum showed two minima near 210

and 220 nm, possibly due to the induction of helical-like

structure (inset, Fig. 3b), which disappeared with further

increase in temperature. This is an interesting observation

as the optimum temperature and pH for the enzyme activity

are 60 �C and pH 10.0.

Different modes of thermal denaturation were observed

under three pH conditions. At extreme acidic pH i.e. at pH

1.0, the negative ellipticity at 212 nm, remained fairly

Fig. 1 Stability of NprotI at

different pH: activity, CD and

fluorescence profiles.

a Functional stability profile at

25 �C. b Far UV CD spectra

(250 lg/ml, 6 h incubation).

c ANS binding to NprotI.

d Near UV CD spectra (800 lg/

ml, 6 h incubation) at pH 1 and

5. Number on each spectrum

indicates pH

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intact at all temperatures. While at pH 10.0 and pH 5.0,

different response was observed. The positive ellipticity at

230 nm decreased under all the pH conditions with

increase in temperature. Tm values were calculated by

sigmoidal fit of the MRE at 230 nm, as, 73 �C at pH 1.0,

71 �C at pH 5.0 [3] and 61 �C at pH 10.0 (Fig. 3c).

3.2.3 Differential Scanning Calorimetry of NProtI

at Different pH

The DSC scans for NprotI were run at different pH. The

scan at pH 5.0, the native pH, showed a Tm of 76.4 �C [3].

At pH 1.0, the apparent Tm of 72.7 �C was observed (Fig

S2) indicating that the entity formed at pH 1.0 is com-

paratively stable. At pH 10.0, the optimum pH for NprotI

activity, the apparent Tm (63.8 �C) was much lower than

that at pH 5.0 and pH 1.0. The increase in enthalpy and a

decrease in Tm could be due to change in structure of the

protein. The data is presented in Table 1.

There is noticeable difference between the Tm values

determined by CD spectroscopy and DSC at pH 5.0. This

could be due to the scan rate dependence which is the

property of the kinetically stable proteins. This phenome-

non is prominent at pH 5.0, where it is highly stable in

presence of GdnHCl [3].

When the enzyme was incubated with PMSF prior to

DSC scan, the Tm value was slightly increased from 76.4 to

78.2 �C indicating modification or blocking of the active

site serine residue increases the stability of the protein.

3.3 Effect of GdnHCl on NprotI at pH 1.0

The ability of NprotI (at pH5.0) to resist chemical dena-

turation even at high concentrations of denaturants, e.g.,

6 M GdnHCl and the role of the special structural feature

i.e. PPII fold therein have been established [3]. Here, the

protein stability at pH 1.0 in presence of GdnHCl has been

investigated (Fig. 4a). Unfolding of the protein at pH 1.0

was observed at C3.0 M GdnHCl after incubation for 24 h

as indicated by a red shift in the kmax from 353 nm to

358 nm. Such a change in the emission kmax was not seen

at pH 5.0 even after 48 h of incubation in the vicinity of

6.0 M GdnHCl [3].

Fig. 2 Stability profile of

NprotI in acidic pH range at

temperatures. a 50 �C and

b 60 �C. Experiment was

performed as described in Sect.

2. Number on each line

indicates pH

Fig. 3 Comparison of Far UV CD spectra of thermal denaturation of

NprotI at pH. a 1.0, b 10.0 Inset of b shows helical like structure

induction at 65 �C, pH 10.0. c Denaturation curves based on

MRE230 nm. The enzyme was incubated at respective pH for 4 h

before monitoring the thermal transitions. The enzyme was then

subjected to increasing temperature with incubation time of 10 min at

each temperature before recording the CD scans

64 S. Rohamare et al.

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3.4 Effect of Guanidine Thiocyanate on Structure

and Activity of NprotI

As NprotI was resistant to GdnHCl at pH 5.0 [3], in the

present work, GuSCN, the denaturant was used to unfold

the enzyme. As seen in the activity profile (Fig S3), the

activity of NprotI increases in the presence of low con-

centrations of GuSCN, whereas the activity decreased by

50 % upon incubation for 20 min at 6 M concentration.

Incubation with 5 M GuSCN for 24 h led to about 90 %

loss of activity. In the presence of 2 M GuSCN, about

50 % activity was still retained even after 7 days.

Interestingly; the fluorescence studies in presence of

GuSCN showed blue shift in kmax at lower concentration,

with concomitant increase in activity (Fig. 4b). As far as

chemical denaturation is considered, NprotI could be

denatured only with GuSCN at pH 5.0 and with GdnHCl at

pH 1.0.

4 Discussion

NprotI, a serine protease from Nocardiopsis sp. exhibits

optimum activity at pH 10.0, but is most stable at pH 5.0

[3, 6]. The enzyme possesses PPII fold and exhibits

unusual stability in presence of denaturing agents such as

GdnHCl (24 h in 6 M), and organic solvents like methanol,

ethanol and DMSO (dimethyl sulfoxide) (all 75 % v/v for

24 h), as well as in proteolytic environment [6]. In the

present work, we have investigated the functional and

structural stability of NprotI over a wide range of pH for

extended time period. Also, a correlation between thermal

stability at different pH with special emphasis on PPII fold

has been investigated. Differential modes of structure–

function relationship were observed at acidic and alkaline

pH. The results have been summarized in Table 2.

4.1 Functional and Structural Stability of NprotI

at Different pH

In spite of being an alkaline enzyme, NprotI was found to

be more stable and showed enhanced activity when incu-

bated at acidic pH, while drastic loss in the activity was

observed after incubation at pH 10.0 in absence of sub-

strate. Although at pH 10.0 the protein is labile, the con-

formation is favorable for catalysis if substrate is provided.

The loss in activity was not reflected much in the sec-

ondary structure, as NprotI maintained the overall PPII fold

in broad pH range. A trough rather than sharp minimum

was observed at pH 10.0 in far UV CD spectrum. A similar

shift in the minima by 4 nm in b—casein was observed at

alkaline pH (10.5) compared to that at pH 6.75 [7].

Other kinetically stable proteins like milin and Nocar-

diopsis alba protease (Napase) were also found to be stable

over a wide range of pH. Napase has been very well studied

for the high acid stability which was attributed to the

reorientation of surface charge residues [4]. Milin was

reported to be stable due to heavy glycosylation [2].

Among the very few reports available on correlation of

PPII fold transitions with pH, Rucker et al. [8] have

reported the effect of pH on homopolymer lysine. The

lysine peptide maintained its PPII structure in varied pH

conditions even when side chain charges were heavily

screened or neutralized. The plausible explanation given

Table 1 Summary of the analysis of DSC profile of NprotI at dif-

ferent pH

pH Tm(�C) DSC DH(kcal/mol)

1 72.7 140

2 75.7 119

3 74.7 140

5 76.4 90

7 74.0 268

10 63.8 499

5, PMSF 78.2 117

Fig. 4 a Denaturation profile of

NprotI treated with 0–6 M

GdnHCl, at 25 �C, for 24 h. pH

1 (filled squares); pH 5(empty

squares). b Fluorescence (empty

squares) and activity (filled

squares) based denaturation

profile of NprotI incubated in

GuSCN (0-6 M) at 25 �C

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was that the peptide backbone favored the PPII structure to

have favorable interaction with solvent. In another study

[9], type IV collagen was reported to maintain its PPII fold

at acidic as well as neutral pH.

Although we have studied the structural stability of PPII

fold of NprotI over a wide pH range, there was no corre-

lation of this structural stability with functional stability of

the enzyme (which was clearly seen during GdnHCl

treatment profile [3]). This could be possibly due to

changes in the charged state of active site amino acid

residues at different pH.

4.2 Molten Globule Like Structure of NprotI

Retention of secondary structure and exposed hydrophobic

amino acids in NprotI at pH 1.0 indicated possible for-

mation of molten globule structure of the molecule. The

common molten globule conformation reported in the lit-

erature [10] is a partly folded one. It has less secondary

structure, but is significantly compact as the native con-

formation. Also, it has more hydrated hydrophobic residues

and apparently has no defined tertiary structure [11]. The

CD data presented suggests that at pH 1.0, NprotI could

adopt a molten globule like structure with stabilized ter-

tiary interactions, the fact which could be responsible for

the retention of the active structure. Protonation at lower

pH confers unusual properties to NprotI. The structure at

pH 1.0 has stable tertiary structure with few hydrophobic

side chains exposed. It showed enhanced catalytic rate

when checked under standard assay conditions. The

immunoglobulin MAK33 at acidic pH assumes a confor-

mation which appears to be stabilized by tertiary interac-

tions in addition to possessing all the characteristics of a

molten globule [12]. Refolded conformation (A-state) at

pH 1.4 was observed for glucose oxidase showing 90 % of

native secondary structure and native-like near-UV CD

spectral features [13].

4.3 Thermostability of NprotI at Different pH

Further enhancement in the activity of NprotI at pH 1.0–3.0

at higher temperature was an interesting observation.

Going towards extreme acidic side from pH 3.0 to 1.0, the

stability (and enhancement in activity) temperature

decreased from 60 to 50 �C. The exposed hydrophobic

patches at pH 1.0 are more than those at pH 3.0, as indi-

cated by ANS binding. This could be the probable reason

for different response observed at 50 and 60 �C. The higher

rate of catalysis at acidic pH and loss of activity at alkaline

pH; both at higher temperature, could be correlated with

the structural transitions monitored by CD spectroscopy.

The PPII fold of NprotI incubated at pH 1.0 remained fairly

intact up to 70 �C. Based on the isodichroic point and Tm

values revealed by secondary structural transitions, differ-

ent modes of thermal denaturation at pH 1.0, 5.0 and 10.0

were observed. Lower Tm value at pH 10.0 as compared to

the Tm values at pH 1.0 and pH 5.0 were obtained from

both CD spectroscopy and DSC studies. Retention of

Table 2 Summary of structure–function profile of NprotI

pH 5.0 (standard) pH 1.0 pH 3.0 pH 10.0

Optimum activity – – – ???

Residual activity after

incubation of enzyme alone

?(at 25 �C)

?(at 50� and 60 �C)

??(at 25 �C)

???(at 50 �C)

?(at 60 �C)

??(at 25 �C)

???(at 50 �C)

????(at 60 �C)

-(at 25 �C)

Total loss

Intrinsic fluorescence

(intensity and kmax)

353 nm Both unchanged Both unchanged Slightly reduced and

kmax unchanged

ANS binding ? ??? ?? –

Far UV CD (maximum at

230 nm unchanged)

Standard profile

(Fig. 1b)

Minimum at 212 nm

sharpens and

ellipticity reduced

Minimum at 212

sharpens and ellipticity

slightly reduced

Minimum at 212 nm

becomes shallow

Near UV CD Standard profile

(Fig. 1d)

Similar to standard NA NA

Thermal unfolding, Tm (far

UV CD)

71 �C 73.7 �C NA 61.4 �C

DSC, Tm 76.4 �C 72.7 �C 74.7 �C 63.8 �C

GuSCN Stable in 2 M

up to 7 days

NA NA NA

Gdn-HCl Stable in 6 M

for 7 days

Stable in 2 M for 24 h NA NA

NA not applicable

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functional activity after incubation at low pH is supported

by structural integrity of the protein as indicated by higher

thermostability of the acid induced state.

A similar trend was observed for thermolysin, a neutral

metalloproteinase from Bacillus thermoproteolyticus rokko

[14]. For thermolysin, the enthalpy value increased and Tm

decreased at pH 9.0 as compared to pH 5.0. Possible de-

protonation of amino acid side chains of NprotI at pH 10.0

could lead to changes in the active site geometry leading to

most favorable structure for catalysis. pORF2 (protein from

Open Reading Frame 2) of Hepatitis E Virus [15] was

reported to acquired more secondary structure and exhibit

higher thermal stability at acidic pH, which could be due to

the presence of one or more pair(s) of carboxylate groups

which would prevent destabilization due to repulsion

between positive charges. For NAPase, a novel mechanism

of acid resistance through charge migration has been pro-

posed based on structural and mutational studies [4].

Fink et al. [16] have classified acid denatured states of

proteins in three major types. For type I proteins two

transitions were seen, when titrated with HCl in the

absence of salts. In the vicinity of pH 3–4, initially

unfolding was observed and then refolding to the A state,

i.e. a molten globule-like conformation, at lower pH. The

type II proteins were not seen to fully unfold upon acid

titration but they directly transformed into the molten

globule state, typically in the vicinity of pH 3.0. No sig-

nificant unfolding at pH as low as 1.0 was observed in case

of type III proteins, but they may behave similarly to type I

in the presence of urea. Based on our observations, NprotI

could be of type III protein. Other proteins of this type are

chicken lysozyme, ubiquitin, protein A, T4 lysozyme,

chymotrypsinogen, lactoglobulin and conA [16].

4.4 Stability of NprotI Towards Chemical Denaturants

While, NprotI was found to be stable towards GdnHCl at pH

5.0, it was found to be labile to denaturation by GuSCN at pH

5.0 and by GdnHCl at pH 1.0. But, the protein was found to be

equally thermostable at pH 1.0 and at pH 5.0 as monitored by

CD spectroscopy. Also, activity studies showed the protein to

be more stable at acidic pH at higher temperature. All these

observations suggested stability of NprotI towards physical

denaturants at pH 1.0 and instability towards chemical dena-

turants. This was supported by the observation that at pH 1.0,

NprotI got denatured in presence of potassium iodide (a

negatively charged quencher) during quenching studies [17].

This indicated importance of electrostatic interactions (which

could be getting affected during chemical denaturation at pH

1.0) in holding the conformation of the protein.

To summarize, the conformation and function of NprotI

is highly stable at extreme acidic pH, a property which

makes this enzyme a good candidate for further studies.

Acknowledgments The authors thank Dr. M. Fernandes, Organic

Chemistry Division, NCL, for allowing the use of CD spectropolar-

imeter. SR, SD and PKN were supported by Senior Research Fel-

lowships from the CSIR, New Delhi, India. The MicroCal VP-DSC

equipment used in this study was supported by a UPE grant from the

UGC (India) to the University of Hyderabad.

References

1. Cunningham EL, Jaswal SS, Sohl JL, Agard DA (1999) Kinetic

stability as a mechanism for protease longevity. Proc Natl Acad

Sci 96:11008–11014

2. Yadav SC, Jagannadham MV (2009) Complete conformational

stability of kinetically stable dimeric serine protease milin against

pH, temperature, urea, and proteolysis. Eur Biophys J 38:981–990

3. Rohamare SB, Dixit VS, Nareddy PK, Sivaramakrishna D,

Swamy MJ, Gaikwad SM (2013) Polyproline fold—in imparting

kinetic stability to an alkaline serine endopeptidase. Biochim

Biophys Acta 1834:708–716

4. Kelch BA, Eagen KP, Erciyas FP, Humphris EL, Thomason AR,

Mitsuiki S, Agard DA (2007) Structural and mechanistic explo-

ration of acid resistance: kinetic stability facilitates evolution of

extremophilic behavior. J Mol Biol 368:870–883

5. Matthew JB (1985) Electrostatic effects in proteins. Annu Rev

Biophys Chem 14:387–417

6. Dixit VS, Pant A (2000) Comparative characterization of two

serine endopeptidases from Nocardiopsis sp. NCIM 5124. Bio-

chim Biophys Acta 1523:261–268

7. Qi PX, Wickham ED, Farrell HM Jr (2004) Thermal and alkaline

denaturation of bovine b-casein. Protein J 23:389–402

8. Rucker AL, Creamer TP (2002) Polyproline II helical structure in

protein unfolded states: lysine peptides revisited. Protein Sci

11:980–985

9. Shimizu A, Kawai K, Yanagino M, Wakiyama T, Machida M,

Kameyama K, Naito Z (2007) Characteristics of type IV collagen

unfolding under various pH conditions as a model of pathological

disorder in tissue. J Biochem 142:33–40

10. Ptitsyn OB (1987) Protein folding: hypotheses and experiments.

J Protein Chem 6:277–293

11. Ewbank JJ, Creighton TE (1991) The molten globule protein

conformation probed by disulphide bonds. Nature 350:518–520

12. Buchner J, Renner M, Lilie H, Hinz HJ, Jaenicke R, Kiefhaber T,

Rudolph R (1991) Alternatively folded states of an immuno-

globulin. Biochemistry 30:6922–6929

13. Haq SK, Ahmad MF, Khan RH (2003) The acid-induced state of

glucose oxidase exists as a compact folded intermediate. Bio-

chem Biophys Res Commun 303:685–692

14. Conejero-Lara F, Azuaga AI, Mateo PL (1997) Differential

scanning calorimetry of thermolysin and its 255–316 and

205–316 C-terminal fragments. React Funct Polym 34:113–120

15. Zafrullah M, Khursheed Z, Yadav S, Sahgal D, Jameel S, Ahmad

F (2004) Acidic pH enhances structure and structural stability of

the capsid protein of hepatitis E virus. Biochem Biophys Res

Commun 313:67–73

16. Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR (1994)

Classification of acid denaturation of proteins: intermediates and

unfolded states. Biochemistry 33:12504–12511

17. Rohamare SB, Gaikwad SM (2014) Tryptophan environment and

functional characterization of a kinetically stable serine protease

containing a polyproline II fold. J Fluoresc 24:1363–1370

Kinetically Stable Alkaline Serine Protease Possessing Polyproline II Fold 67

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