Báo cáo khoa học: Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes ppt

Chemically stabilizing osmolytes low molecular mass organic compounds that raise the midpoint of thermal denaturation are divided into three classes: amino Keywords catalytic efficiency;

stability in the presence of all classes of stabilizing

osmolytes

Shazia Jamal*, Nitesh K Poddar*, Laishram R Singh*,, Tanveer A Dar*,, Vikas Rishi§ and Faizan Ahmad

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India

Introduction

Both prokaryotic and eukaryotic cells, when subjected

to harsh environmental conditions such as water, salts,

cold and heat stresses, adopt a common strategy in

protecting their proteins by producing low molecular

weight organic substances called osmolytes [1,2] Chemically stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) are divided into three classes: amino

Keywords

catalytic efficiency; denaturation equilibrium;

enzyme activity; osmolytes, protein

stability

Correspondence

F Ahmad, Centre for Interdisciplinary

Research in Basic Sciences, Jamia Millia

Islamia, New Delhi, India 110025

Fax: +91 11 2698 3409

Tel: +91 11 2698 1733

E-mail: faizan_ahmad@yahoo.com

*These authors contributed equally to this

work

Present addresses

Division of Population Science, Fox

Chase Cancer Center, Philadelphia, PA,

USA

Department of Chemistry Biochemistry,

University of Montana, Missoula, MT,

USA

§National Cancer Institute, NIH, Bethesda,

MD, USA

(Received 29 May 2009, revised 10 August

2009, accepted 19 August 2009)

doi:10.1111/j.1742-4658.2009.07317.x

We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stabil-ity and function of RNase-A under physiological conditions (pH 6.0 and

25C) Measurements of Gibbs free energy change at 25 C (DGD) and kinetic parameters, Michaelis constant (Km) and catalytic constant (kcat) of the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability (a) Polyhydric alcohols and amino acids and their derivatives do not have significant effects on

DGD and functional activity (Km and kcat) (b) Methylamines increase

DGD and kcat, but decrease Km (c) Sugars increase DGD, but decrease both Kmand kcat These findings suggest that, among the stabilizing osmo-lytes, (a) polyols, amino acids and amino acid derivatives are compatible solutes in terms of both stability and function, (b) methylamines are the best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but they apparently do not yield functionally active folded molecules

Abbreviations

acids and their derivatives, polyhydric alcohols and

sugars, and methyl ammonium derivatives [1] These

osmolytes are known not only to stabilize proteins

[3,4], but they also induce refolding of misfolded

proteins [5–8] and remove protein aggregation [9–12]

Mechanisms of protein osmolyte interactions, the

effect of osmolytes on protein stability, and how

osmo-lytes correct protein misfolding defects and remove

protein aggregation have been widely investigated

It has been demonstrated that the unfavourable

interaction between the peptide backbone and the

osmolytes leading to the preferential hydration of the

protein domain is the driving force of protein

stabiliza-tion or folding [3,4] Furthermore, the effect of

osmolytes on the functional activity of an enzyme has

also been investigated on a number of enzymes

Conse-quently, this has led to the classification of osmolytes

into two classes: compatible or counteracting

Compat-ible osmolytes increase protein stability against

denaturation with little or no effect on their function

under native conditions [1,13,14] Representatives of

this class include certain amino acids (e.g proline

and glycine) and polyols (e.g trehalose, sucrose and

sorbitol) Counteracting osmolytes consist of the

methylamine class of osmolytes, which are believed to

have the special ability to protect intracellular

proteins against the inactivation⁄ destabilization by

urea [14–17] In contrast to compatible osmolytes,

counteracting osmolytes are believed to cause changes

in protein function that are opposite to the effects that

urea has on protein function [16–19]

Despite significant advances in understanding the

effect of osmolytes on protein stability, folding and the

activity of proteins and enzymes, the relationship

between protein stabilization by osmolytes and its

con-sequent effects on the activity of enzymes has not been

examined It is not yet understood how well protein

stability and activity are coupled in the presence of an

osmolyte This study was undertaken to investigate the

relationship between protein stability and activity

changes in the presence of a wide range of osmolytes

For this we evaluated the protein stability (DGD,

Gibbs free energy change at 25C) of RNase-A and

its activity parameters (Km, Michaelis constant; kcat,

catalytic constant) in the presence and absence of

almost all naturally occurring osmolytes We report

here that protein stability and activity are not largely

coupled in the presence of osmolytes However, protein

stability and activity have a linear correlation in the

presence of methylamines and sugar osmolytes This

study, in fact, has led to the classification of osmolytes

into three different classes based on their effects on

stability and activity parameters of RNase-A

Results and Discussion Protein stability and enzyme activity have a well-corre-lated function However, we do not know how this relationship is maintained in the presence of stabilizing osmolytes accumulated under stressed conditions Because stabilizing osmolytes do not have a direct interaction with the protein domains per se, it is expected that an increase in protein stability (DGD) by

an osmolyte due to the shift in the denaturation equi-librium, native state M denatured state, towards the left, must increase the catalytic efficiency of the enzyme and vice versa The reason for saying this is that urea, which decreases DGD, is known to decrease the cata-lytic efficiency of osmolytes [20, references therein] Thus, it will be interesting to investigate how kinetic parameters of the enzyme-catalyzed reaction change upon modulation of protein stability (DGD) by osmo-lytes To investigate the protein stability–activity rela-tionship in the presence of osmolytes, we intentionally chose two different groups of osmolytes The first group consists of polyols, amino acids and amino acid derivatives, which have been reported to have no effect

on DGD associated with the protein denaturation equilibrium, native state M denatured state, under physiological conditions The second group consists of methylamines and sugars, which are shown to increase

DGD of proteins associated with the denaturation equilibrium, native state M denatured state The observed effects of polyols, sugars and methylamines and some amino acids on DGD of RNase-A have been reported previously [21–25], and DGD values in the presence of these osmolytes are given in Table 1 How-ever, DGD values of RNase-A in the presence of alanine, serine, lysine, b-alanine, taurine and dimethyl-glycine have not been published elsewhere We have therefore measured the thermodynamic parameters of RNase-A in the presence of these amino acids and amino acid derivatives, and values of DGD, measured

in triplicate, are given in Table 1

The effect of polyols on the kinetic parameters (Km and kcat) of the RNase-A mediated hydrolysis of cyti-dine 2¢-3¢ cyclic monophosphate has been previously reported [22] Values of the kinetic parameters of this protein in the presence of all other osmolytes were determined and are presented in Table 1 It should be noted that the value for each kinetic parameter repre-sents the mean of three independent measurements together with the mean error These kinetic parameters

in the absence of the osmolytes, shown in Table 1, are

in excellent agreement with those reported previously [26–28] These agreements led us to believe that our measurements of the enzyme-catalyzed reactions and

[Sugars] M

1 )

kcat )(s

1 )

Km (m

[Polyols] M

1 )

k cat )(s

1 )

Km (m

[Amino acids

derivatives] M

1 )

kcat )(s

1 )

Km (m

[Methylamines] M

1 )

kcat )(s

1 )

Km (m

Taurine 0.25

the analysis of the progress curves for kinetic

parame-ters are accurate It can be seen in Fig 1 (see also

Table 1) that sugars and methylamines affect both the

thermodynamic (DGD) and the kinetic (Km and kcat)

properties, whereas polyols, amino acids and amino

acid derivatives do not have any significant effect on

these parameters In fact, based on the effects that the

osmolytes have on both DGD and the catalytic

prop-erties of RNase-A (Table 1), we can distinctly classify

osmolytes into three different classes (a) Class I

includes polyhydric alcohols (sorbitol, glycerol, xylitol,

adonitol, mannitol) and amino acids and derivatives

(glycine, alanine, proline, serine, lysine, b-alanine and

taurine) that have no significant effects on both DGD

and kcat (b) Class II represents methylamines

(sarco-sine, dimethylglycine, betaine, trimethylamine N-oxide) that increase both DGD and kcat, but decrease Km (c) Sugars (glucose, fructose, galactose, sucrose, raffinose, stachyose) that increase DGD, but decrease both Km

and kcatbelong to class III

kcatalone does not absolutely define the overall cata-lytic activity of an enzyme, as it is a first-order rate constant that refers to the properties and reactions of the enzyme–substrate, enzyme–intermediate and enzyme–product complexes [29] On the other hand,

kcat⁄ Kmis an apparent second-order rate constant that refers to the properties and the reaction of the free enzyme and free substrate [29] We have therefore esti-mated kcat⁄ Km values of all the reactions in the pres-ence and abspres-ence of all classes of osmolytes It can be

versus [osmolyte] (right panels).

seen in Fig 1 that class I osmolytes (polyhydric

alco-hols, amino acids and amino acid derivatives) do not

significantly perturb kinetic parameters (Km and kcat)

and, hence, the overall catalytic efficiency (kcat⁄ Km) of

RNase-A This observation on the effect of polyols

and amino acids on RNase-A is in agreement with that

on other enzymes (lactate dehydrogenase, lysozyme,

pyruvate kinases) reported previously [13,22,30] It has

been argued that these compatible osmolytes affect the

association of the substrate with the enzyme in any

one of several ways, e.g through solvation effects on

substrates or enzyme active sites and through their

effects on the thermodynamic activity of substrates

and enzymes [13,30,31] Thus, a lack of effect on both

enzymatic parameters (Km and kcat) of RNase-A

sug-gests that polyols, amino acids and amino acid

deriva-tives have little or no effect on the solvation properties

of the substrate and the enzyme active sites or on their

thermodynamic activities Another explanation for

these observations comes from our DGD

measure-ments Because of perfect enthalpy–entropy

compensa-tion, DGD is unperturbed in the presence of class I

osmolytes (see Table 1), i.e the denaturation

equilib-rium, native state M denatured state, of RNase-A is

unperturbed and, hence, no change in the functional

activity of the enzyme in the presence of such

osmo-lytes (see Fig 1)

If our explanation is correct, an increase in protein

stability (DGD) by osmolytes must result in an

increase in the number of N molecules due to a shift

in the denaturation equilibrium, native state M

dena-tured state, towards the left Consequently, both kcat

and kcat⁄ Kmare expected to increase in the presence of

such osmolytes, as kcat⁄ Km refers to the reaction of

free (active) enzyme [29] Data presented in Table 1

and Fig 2 for the effect of methylamines (class II) on

DGD and kinetic parameters show that this is indeed

true It is noteworthy that our observation of the effect

of methylamines on RNase-A is also in agreement with

previous reports on many other enzymes, such as

rab-bit muscle lactate dehydrogenase, triose phosphate

isomerase, pyruvate kinase, creatine kinase, A4-lactate

dehydrogenase, glutamate dehydrogenase,

argininosuc-cinate lyase, porcine arginosuccinase [17,19,32–35]

However, it should be noted that both Km, the overall

dissociation constant of all enzyme bound species [29],

and kcat are decreased in the presence of sugar (class

III) osmolytes (see Fig 1, Table 1) One possible

explanation for this observation is that the original

native state ensembles and⁄ or the refolded protein

molecules in the presence of sugars undergo a subtle

change in conformation, yielding all or some enzyme

bound species that are more stable than those in the

absence of sugars, i.e Km is decreased On the other hand, this change in conformation results in a decrease

in kcat, the turnover number of the enzyme in the pres-ence of sugars, i.e the maximum number of substrate molecules converted to product per active site per unit time is decreased A subtle change in the enzyme active site that occurs in the presence of sugars may be a pos-sible cause for the observations on Km and kcat of RNase-A in the presence of class III osmolytes

To evaluate if all the refolded protein fractions produced by an osmolyte are in functionally active conformation, we determined the relationship between changes in protein stability (DDGD) and overall catalytic efficiency (Dlog(kcat⁄ Km)) in the presence of

Fig 2 Relationship between protein stability and catalytic

the presence of various osmolytes.

different concentrations of each osmolyte (Fig 2) It

can be seen in Fig 2 that for class I osmolytes

(poly-ols, amino acids and amino acid derivatives) the slope

is nearly 0 This is an expected result, as there is no

perturbation of the denaturation equilibrium and,

hence, there is no increase in catalytic efficiency in the

presence of this group of osmolytes Interestingly,

there is a linear relationship between DDGD and

Dlog(kcat⁄ Km) in the presence of methylamines and

sugar However, the slopes of the plot (Dlog(kcat⁄ Km)

versus DDGD) are very different In fact, the slope in

the presence of sugar osmolytes is 10 times less than

that in the presence of methylamines A higher slope

in the case of methylamines will mean that the total

refolded protein fraction generated by the

methylam-ines is more active than those generated by sugars

Taking these observations and kcatvalues of RNase-A

in the presence of class II and III osmolytes, it seems

that the refolded protein fraction in the presence of

sugars is not as active as the original native molecules,

whereas it is opposite in the presence of methylamines

We therefore conclude that equilibrium shift is not the

only ultimate step to increase the activity of an enzyme

in the presence of osmolytes

In general, two thermodynamic models are used to

explain the effect of osmolytes on protein stability [36,

references therein] The binding model claims that an

increase in the osmolyte-induced stability arises from

the preferential hydration (or exclusion of the

osmo-lyte) leading to a shift in the denaturation equilibrium,

native state M denatured state, towards the left The

excluded volume model focuses on the fact that

osmo-lytes limit the conformational freedom of proteins by

driving them to their most compact native state

(cata-lytically most efficient form) The decrease in

confor-mational freedom arises from steric repulsions between

the protein and the osmolyte The latter model

assumes that the native state of a protein consists of

inter-converting high (most compact) and low (less

compact) activity state ensembles and also

demon-strated that the presence of osmolytes shifts the native

conformational equilibria towards the most compact

protein species within native state ensembles [32,37,38]

The variation in the effect of stabilizing osmolytes in

modulating the catalytic efficiency of RNase-A in the

presence of each class of osmolyte may best be

explained by the combination of both thermodynamic

models Our results suggest that: (a) methylamines not

only decrease conformational freedom, but also

increase preferential hydration, which consequently

generates more active protein molecules; (b) sugar

osmolytes affect the conformational freedom and

preferential hydration in such a way that it produces

catalytically less competent species; and (c) class I osmolytes have no significant effects on both the conformational freedom and the preferential hydration

of the protein In agreement with the explanation

on methylamines, previous reports on trimethylamine N-oxide indicate that it not only produces more active molecules by shifting the denaturation equilibrium [24,25,36,39], but also affects the native state by con-verting the low activity ensembles to the high activity ensembles [37] Very interestingly, a recent refolding kinetic study of carbonic anhydrase II in sucrose showed that the sugar significantly accelerates the rate

of refolding of the enzyme to the native or compact near-native conformations, but decreases the fraction

of catalytically active enzyme recovered [40]

It has already been reported that osmolytes indepen-dently affect proteins and, hence, their effects are algebraically additive [21,41] Based on our results given in Table 1, one can speculate that: (a) the poly-ols–amino acids (or amino acid derivatives) system is

an exclusive mixture that is compatible both with thermodynamic stability (DGD) and function, and (b) sugar–methylamine mixtures are attractive candidates

to yield amazingly enhanced protein stability and function Thus, different osmolyte mixtures may serve

as post-translational modulators of stability and⁄ or function of many enzymes This may perhaps be the main reason why many organisms use multi-osmolyte systems [1,14,15,42–44]

Furthermore, the osmolyte-induced folding of pro-teins is determined by interactions of the osmolyte with all protein groups (peptide backbone and side chains) exposed on denaturation For various osmo-lytes, Bolen & Baskakov [3] have shown that: (a) the main driving force for the folding is the unfavourable interaction between the osmolyte and the peptide back-bone, and (b) the total contribution of side chains to the stability of the native state, which may interact differently with different osmolytes, is very small These conclusions are supported by our measurements

of DGD of RNase-A in the absence and presence of sugars and methylamines It is seen in Table 1 that, on the molar scale, these osmolytes, which are chemically different, have, within experimental errors, almost identical effects on DGD

We are confident of three findings: (a) Polyols, amino acids and amino acid derivatives are ideal osmolytes, for they neither perturb the denaturation equilibrium nor affect the functional activity under native conditions However, they have the ability to protect proteins from denaturing stresses (b) Methyl-amines not only stabilize proteins, but also refold the denatured protein to a more active state under native

conditions (c) Sugar osmolytes stabilize proteins, but

they convert the denatured protein molecule to a less

active form under native conditions These findings

make these chemical chaperones aptly suitable for

structure–function studies of proteins, as each class of

osmolytes (classes I–III) can modulate the stability

and⁄ or function of a protein differently

Experimental procedures

Chemicals

Commercial lyophilized preparations of RNase-A (type

III-A) were purchased from Sigma Chemical Company

(St Louis, MO, USA) d-glucose, d-fructose, d-galactose,

dimethylglycine, glycine betaine, trimethylamine N-oxide,

and cytidine 2¢-3¢ cyclic monophosphate were also obtained

from Sigma These and other chemicals, which were of

analytical grade, were used without further purification

Dialysis and the determination of the

concentration of protein

An RNase-A solution was dialyzed extensively against

filtered using 0.45 lm Millipore filter paper The protein

gave a single band during the native and SDS

poly-acrylamide gel electrophoresis The concentration of the

protein stock solution was determined experimentally using

a value of 9800 at 277.5 nm for e, the molar absorption

measurements were prepared in 0.05 m cacodylic acid buffer

containing 0.1 m KCl Because the pH of the protein

solu-tion may change on the addisolu-tion of the osmolytes, the pH

of each solution was also measured after each measurement

It was observed that the change in pH was not significant

Activity measurements

In order to see the effect of an osmolyte on the kinetic

the enzyme were preincubated in a given concentration of

each osmolyte Following the procedure described

previ-ously [22], RNase-A activity using cytidine 2¢-3¢ cyclic

monophosphate as a substrate was measured The progress

curve for RNase-A mediated hydrolysis of cytidine 2¢-3¢

cyclic monophosphate in the concentration range 0.05–

concentration of each osmolyte was followed by measuring

the change in absorbance at 292 nm for 20 min in a Jasco

Japan) Sample and reference cells were maintained at

sub-strate concentration and in the absence and presence of a fixed osmolyte concentration, initial velocity (m) was deter-mined from the linear portion of the progress curve, usually

30 s The plot of initial velocity (m) versus [S] (in mm) at

using Eqn (1)

Thermal denaturation measurements

Thermal denaturation studies were carried out in a Jasco

Peltier-type temperature controller (ETC-505T), with a heating

increas-ing temperature was followed at 287 nm for RNase-A Approximately 650 data points of each transition curve were collected The raw absorbance data were converted into (De287), the difference molar absorption coefficient

Tm) using a nonlinear least squares analysis according to the relationship described earlier (see equation (1) in [25])

the Gibbs–Helmholtz equation with known values of Tm,

(see equation (2) in [25])

Acknowledgements

FA is grateful to the Department of Science and Technology (India) and the Council of Scientific and Industrial Research (India) for financial support

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