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Preferential Water Exclusion in Protein Unfolding Pulikallu Sashi, U. Mahammad Yasin, Harihar Balasubramanian, M. Usha Sree, Dasari Ramakrishna, and Abani K. Bhuyan* School of Chemistry University of Hyderabad, Hyderabad 500 046, India S Supporting Information *

ABSTRACT: Association of water with protein plays a central role in the latter’s folding, structure acquisition, ligand binding, catalytic reactivity, oligomerization, and crystallization. Because these phenomena are also influenced by the net charge content on the protein, the present study examines the association of water with cytochrome c held at different pH values so as to allow its side chains to ionize to variable extents. Equilibrium unfolding of differently charged cytochrome c molecules in water−methanol binary mixtures, where the alcohol acts as the cosolvent denaturant, was used to quantify the preferential exclusion of water during the unfolding transition. The extent of exclusion was found to be related to the net-charge-dependent molecular expansion of the protein in an alcohol-free aqueous medium. The degree of water exclusion was also found to be linearly related to the observed rate of protein unfolding, where the net charge contents of the initial and final states are the same. The results suggest that side-chain ionization, molecular expansion due to charge repulsion, and hence the loss of tertiary contacts lead to additional water−protein association. Protein unfolding rates appear to be linearly correlated with the effective number of water molecules excluded across the end states of unfolding equilibria.

1. INTRODUCTION Water plays numerous roles in the structure, function, and folding dynamics of proteins. A few intrinsic water molecules, which are lodged in protein cavities and have residence times in the nano- to millisecond range because of their exchange with bulk water, are necessary for the maintenance of the native-state structure, binding, and catalysis.1 Surface waters, which are larger in number, have residence times of tens to a few hundreds of picoseconds.2−4 The residence time of surface water molecules is apparently one of the factors involved in protein stability.5 Surface-bound waters, whose density is simulated to be greater than that of bulk water,6 are thought to be involved in chain collapse and tertiary structure formation during folding.7 Studies on protein−water association are thus centrally important for a complete description of protein phenomena. The extent of interaction of a protein with water molecules, including cavity waters, stoichiometric hydration waters, and any additional waters that might be due to preferential hydration, depends on the nature and activity of a cosolvent, if present. For example, in denaturant-induced protein unfolding where the denaturant is a cosolvent additive, the change in the effective number of water molecules that interact with the protein across the folding−unfolding transition is related to the preferential interaction of the cosolvent with the unfolded chain.8,9 Water−protein association will decrease in the unfolded state if the denaturant preferentially interacts with the unfolded state,10 which is a consequence of the change in the chemical potential of the cosolvent brought about by the protein during unfolding.9 The thermodynamic driving force © 2013 American Chemical Society

for unfolding in the presence of the cosolvent is thus generated by the preferential interaction of the cosolvent with the solventaccessible surface on the protein, thereby influencing protein hydration. The ability of cosolvents to influence protein hydration is often manifested in the chemical reactivity of the protein, its oligomerization, altered ligand affinity, phase transition, and crystallization, for example.11 Regarding the folding−unfolding phase transition, studies in the past have implied preferential binding of cosolvent denaturants such as guanidine hydrochloride (GdnHCl) and urea directly to peptide groups12,13 and aliphatic alcohols to hydrophobic surfaces exposed upon unfolding.14,15 The effective number of water molecules excluded during the native → unfolded transition of cytochrome c due to binding of linear-chain alcohols to the unfolded state has also been quantified.10 However, the response of preferential protein−alcohol interactions, and hence the exclusion of associated water, under conditions where the protein is highly ionized and denatured has not been addressed. Studies of water exclusion in alcohol-induced unfolding of differently charged proteins also provide an opportunity to assess the extent of water−protein interactions affected by net protein charges. With this background, the present study reports on the exclusion of the effective number of cytochrome c-associated water molecules during methanol unfolding of the protein. Water exclusion across the pH scale quantified by methanol titration of cytochrome c clearly Received: November 12, 2013 Published: December 19, 2013 717

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however, because of the presence of four solution components: water, protein, unfolding cosolvent, and destabilizing cosolvent. Considerable simplification is achieved if cosolvent-induced unfolding is carried out under a wide range of pH conditions. Variable protein stability is obtained depending on the pH, and the number of solution components is reduced to three. With this rationale, methanol-induced unfolding of cytochrome c was carried out in the pH range of 2−12. Following Scatchard notation,16 the three solution components are labeled 1, 2, and 3, denoting water, protein, and methanol, respectively. Although the use of variable pH restricts the number of solution components to three, cytochrome c is differently charged and differently folded under different pH conditions. The existence of different pH-specific conformations of cytochrome c (Table 1) appears to suggest different initial

indicates that the number of protein-associated water molecule increases as the net charge on the protein grows and that the number of water molecules excluded scales linearly with the protein unfolding rate.

2. EXPERIMENTAL SECTION 2.1. Methanol Titration of Cytochrome c at Various pH Values. Two stock solutions of precisely 10 μM protein, one in water (native stock) and the other in 60−70% methanol (unfolded stock), were prepared identically using an appropriate buffer (20 mM) for the desired pH. The stock solutions were combined and mixed so as to obtain a series of 0.5 mL samples that differed only in the mole fraction of methanol. This procedure of sample preparation relies on the reversibility of the folding−unfolding reaction and ensured a uniform protein concentration across samples. The samples were allowed to stand for 5−6 h at 23 (±1) °C before their fluorescence emission spectra were measured in the 300−400nm range (excitation at 280 nm). Reported pH values are those read after completion of the experiment. Buffers were glycine− HCl (pH 2−3), sodium acetate (pH 3.5−5.5), 2-[4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, pH 6−7.5), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8−9.5), glycine−NaOH (pH 10−10.5), and 3(cyclohexylamino)-1-propane sulfonic acid (CAPS, 10.5−11.5). 2.2. NMR Measurement of Hydrodynamic Radius (RH). Pulsed-field-gradient (PFG) NMR spectra were recorded as described earlier.10 Although measurement with aqueous protein samples across a pH range of 2−13 was done easily, inclusion of 60−70% CD3OH presented considerable difficulties with protein solubility. Therefore, NMR experiments with methanol-containing samples were limited to fewer pH values. Spectra were recorded at 23 °C on a 500-MHz Avance-III (Bruker) spectrometer and analyzed as published elsewhere.10 2.3. Stopped-Flow Kinetics of Unfolding. About 70 μM aqueous solutions of cytochrome c at different pH values were prepared using the buffer systems mentioned earlier for equilibrium experiments. Solutions were incubated at ∼25 °C overnight. The unfolding solution consisted of 65% methanol in the appropriate buffer (20 mM) whose pH was adjusted to match the pH value of the corresponding initial protein solution to unfold. Unfolding was done by mixing protein and unfolding buffer solutions in a 1:7 ratio in a Biologic SFM-400 instrument whose mixing head was thermostatted at 23 °C. The mixing dead time under the conditions of flow speed and cuvette volume used was ∼1.5 ms. For fluorescence-monitored kinetics, the excitation wavelength was 280 nm, and emission was measured using a 320-nm cutoff filter. At least 10 shots were averaged. Reported pH values are those of waste solutions collected after kinetics runs.

Table 1. pH- and Methanol-Specific Conformational States of Cytochrome c pH

methanol (%)

conformational state

ref

2 2 3 4−10 7 >9 12−13 13 13

0 60 25 0 0 0 0 15−30 70

D (acid denatured) H (alcohol denatured) IM N (native) H (folding intermediate) alkaline UB Ialc (alkaline intermediate) U

17 17 17 17 18 19 20 10 10

states of the protein used for unfolding (N ⇌ U), especially at extremes of pH. The problem could be compounded by suggestions of the presence of equilibrium folding intermediates,18,21,22 presumably in a pH-dependent manner. Misfolding of oxidized cytochrome c due to binding of non-native H26/33 and lysine ligands to the heme iron of the denatured protein can give rise to in additional problem under neutral to basic conditions. These possibilities did not pose serious threats here, however, because this study sought to quantify preferential hydration across the N ⇌ U equilibrium in terms of the energetic and conformational stability of the protein. Protein destabilization at extremes of pH is mainly due to side-chain ionization, which disrupts tertiary structures and expands the molecule through Coulombic repulsion. Most secondary structures more or less prevail under these conditions, although they are weakened.17 Thus, the initial end state of the unfolding equilibrium under highly acidic or basic conditions is a lessstructured and stability-degraded “native” state. It is noted that stability degradation comes at the expense of protein deionization. Hence, stability and charge are mutually inclusive, and analyses and discussions of the methanol-modulated N ⇌ U equilibrium at different pH values must project both. It is also noted that the occurrence of folding intermediates particularly does not need to be considered when water exclusion is quantified across the end states of the N ⇌ U equilibrium alone, because the net number of water molecules expelled presumably reflects the sum of the numbers expelled in different steps. However, the heme mis-ligation problem cannot be averted, and the equilibrium titration data under neutral to basic conditions should be taken to reflect an average of chain configurational heterogeneity in the unfolded state, where the heterogeneity is established by rapid binding and dissociation of both native and non-native intrachain ligands.23

3. RESULTS AND DISCUSSION 3.1. pH-Dependent Stability, Conformation, and Net Charge of Cytochrome c. A basic objective of this study was to determine whether the number of water molecules excluded during unfolding depends on energetic and conformational stability of the protein. Such an investigation could involve a series of cosolvent-induced equilibrium unfolding tests of the protein already destabilized to different extents by the use of low concentrations of another cosolvent, for example, guanidinium-induced unfolding in the presence of low concentrations of urea. Analyses of these data are problematic, 718

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⎛ ∂ ln K ⎞ ⎛ ∂m ⎞U ⎛ ∂m ⎞N − ⎜ 1⎟ ⎜ ⎟ = Δζ1 = ⎜ 1 ⎟ ⎝ ∂ ln a1 ⎠ ⎝ ∂m2 ⎠T , P , μ ⎝ ∂m2 ⎠T , P , μ

3.2. Water Exclusion during Unfolding Is Highly Dependent on Medium pH. Based on the rationales above, we obtained data for methanol-induced equilibrium unfolding of cytochrome c at a number of pH values from 2 to 13 (Supporting Information, Figure S1). Unfolding transitions at extremes of pH lack the initial-state baseline because of severe protein destabilization under these conditions. The transitions were analyzed without invoking equilibrium intermediates (N ⇌ U), and the equilibrium constants K were extracted as a function of water activity. Values of K in Figure 1a are shown in terms of the activity of water in the

1

1

(1)

where Δξ1 is the difference in the preferential interaction of water with the native and unfolded states; m1 and m2 are molal concentrations of water and protein, respectively; and μ1 is the chemical potential of water. The negative ∂ ln K/∂ ln a1 gradient (Figure 1a) indicates that the number of water molecules associated with the protein is larger in the native state, implying a net loss of water molecules (negative Δξ1) when the structure unfolds in the presence of the cosolvent methanol. The variation of Δξ1 with pH reveals that water exclusion during unfolding is a minimum near pH 8 and increases on either side symmetrically by as much as 7-fold in highly acidic and basic media (Figure 1b). Water exclusion during the unfolding reaction is due to the preferential interaction of methanol with exposed hydrophobic surfaces in the unfolded protein that reduces protein−water contacts.10 The point of interest here is the variation in the number of excluded water molecules with pH. Because the change in preferential hydration between the native and unfolded states (eq 1) is a measure of the perturbation of the methanol chemical potential by the protein in the water− protein−methanol system,9 the observed pH dependence of water exclusion (Figure 1b) should arise from a variable binding affinity of methanol toward the unfolded protein. This argument can be extended by considering the chemical potential of methanol (μ3) across the folding−unfolding equilibrium of the protein. The basic principle of phase equilibria requires that μ3 must be the same in the native and unfolded phases across the protein melt, that is, μ3,N = μ3,U. It follows that ° + RT ln C N = μ3,U ° + RT ln C U μ3,N

(2)

where μ3,N ° and μ3,U ° are standard-state chemical potentials of methanol in the native and unfolded phases of the protein, and CN and CU are the corresponding molar concentrations. Equation 2 provides for the partition coefficient KP =

⎡ (μ ° − μ ° ) ⎤ CN 3,N 3,U ⎥ = exp⎢ − ⎢⎣ ⎥⎦ CU RT

(3)

which determines the disparity in the chemical potential of methanol in the native and unfolded phases: the larger the value of μ3,N ° − μ3,U ° , the more strongly methanol binds to the unfolded protein, and therefore, the larger the water exclusion. The dependence of Δξ1 across the pH scale (Figure 1b) then would appear to arise from the variation of KP with the net charge content on the protein (Figure 1c). One can consider the standard free energy of transfer, ΔG°, of the methanol binding equilibrium (CH3OH + protein ⇌ CH3OH−protein) across two pH values, pH(1) and pH(2). Each pH is associated with the equilibrium constant

Figure 1. Methanol-induced unfolding of cytochrome c at different pH values. (a) Dependence of the logarithm of the equilibrium constant, K = U/N, on the activity of water in water−methanol binary mixtures at different pH values: pH 3 (black), pH 4 (red), pH 5 (green), pH 7 (yellow), pH 8 (blue), pH 9 (pink), pH 10 (teal), pH 11 (gray), and pH 12 (maroon). The slopes provide the effective numbers of water molecules excluded, Δξ1, according to eq 1. (b) Variation of Δξ1 with pH. A negative sign for Δξ1 indicates water exclusion. The solid line through the data represents a parabolic fit to the data. (c) Net charge on cytochrome c across the pH scale.

water−methanol binary solvent system, because the objective was to quantify the number of water molecules excluded during unfolding. As discussed earlier,10 the net number of excluded water molecules was obtained using the theory of Gekko and Timashesff, Timashesff, Wyman, and Tanford,8,9,24,25 according to which

K=

aCH3OH−protein aCH3OHa protein

(4)

where the activity is a = mγ and γ is the activity coefficient at unit concentration. The expression for the standard free energy of transfer can now be cast in terms of the equilibrium constants at pH(1) and pH(2) 719

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preferential water exclusion at higher and lower pH values (Figure 1b), the hydrodynamic radius (RH) of cytochrome c across the pH scale was determined by pulsed-field-gradient NMR spectroscopy (Figure 2). Such experiments with

ΔG° = G°(1) − G°(2) = 2.303RT log(K1 − K 2) = 2.303RT (pK 2 − pK1)

(5)

as well as the activities in the solvent systems at pH(1) and pH(2), ΔG° = G°(1) − G°(2) ⎡ a(1) ⎤ CH3OH−protein a(2)CH3OH a(2)protein ⎥ = 2.303RT log⎢ ⎢⎣ a(2)CH3OH−protein a(1)CH3OH a(1)protein ⎥⎦ (6)

so that ΔpK =

a(1)CH3OH−protein a(2)CH3OH a(2)protein a(2)CH3OH−protein a(1)CH3OH a(1)protein

(7) Figure 2. Hydrodynamic radius of cytochrome c in the absence (black) and presence (green) of 75% CD3OH at different pH values. Data near pH 2 shown by dark red symbols are ⟨RH⟩ values of cytochrome c unfolded in 4.5 M urea.

The logarithm of the ratio a(1)/a(2) = mγ(1)/mγ(2) is often referred to as the transfer activity coefficient.26 Therefore, differences in standard-state chemical potentials (μ° = μ − RT ln a) of both methanol and protein at two pH values determine ΔpK and, hence, a difference in the binding constant for the interaction of methanol and protein. 3.3. Difficulties at the Molecular Level. The question of how, at the molecular level, preferential binding of an uncharged solute such as methanol would grow stronger when the net charge content of the protein increases is not straightforward. At a given pH, the net charges of the folded and unfolded states of the protein across the phase equilibrium are the same, but the charge densities are not, which is due to differences in both the charge dispositions and the chain dimensions. In folded conformations, say, when the molar volume of the solute is low, surface charges are favorably watersolvated, and buried charges are generally accommodated in pockets of polar surroundings in the low-dielectric protein interior, which minimizes the energetic cost of their burial. In the unfolded chain, such dispositions of charges are lost, and a chain-contour-length-based charge density, β, can be assigned27 nze β= (8) L

methanol-unfolded protein are challenging, both because the protein precipitates at concentrations higher than ∼90 μM and because the −OH resonance due to 75% CD3OH overwhelmingly dominates the spectrum. As such, the ⟨RH⟩ value of methanol-unfolded protein could be obtained to adequate accuracy for only three pH values. In the absence of methanol, ⟨RH⟩ increases from 17 ± 1 Å in the neutral-pH region to 22 ± 0.5 Å at pH 13 and 30 ± 1 Å at pH 2. This variation arises primarily from side-chain ionization as the protein is placed in increasingly acidic or basic milieus, leading to variable loss of tertiary structure and compactness. Such responses of proteins to acidic and basic media have been widely studied.36−40 The loss of tertiary structure or intraprotein contacts produces two independent effects: The molecule expands due to electrostatic repulsion when solvent counterions are absent, and additional protein−water contacts are established commensurate with the loss of intraprotein contacts. The latter effect is in accordance with the solvent contact model,41 according to which the extent of inter-side-chain contact is inversely proportional to the extent of protein−water contact, assuming no interprotein interactions. Additional water−protein contacts under acidic and basic conditions need not be construed as contributing to the solvation shell of the protein. Indeed, as Timasheff emphasizes,9 one can simply take them as additional proteinassociated waters. Charge-dependent expansion of cytochrome c, and hence an increase in ⟨RH⟩, is observed in the unfolded state as well (Figure 2). Unfolding in the presence of methanol, however, leads to preferential exclusion of water, and the number of excluded water molecules is projected according to the waterassociated status of the protein in the absence of methanol. The pH dependence of Δξ1 (Figure 1b) is a manifestation of this response of the protein. 3.5. Dependence of the Protein Unfolding Rate on the Extent of Water−Protein Association. Because water plays a significant role in protein folding,7 it is desirable to examine the effect of variable water−protein association on folding and unfolding rates at different pH values in such a way that the initial and final pH values of unfolding are held constant. The unfolding experiments described here hold the

where n is the number of charged groups of valence z, e is a unit charge, and L is the end-to-end distance of the chain. Unscreened charges on the protein surface now experience severe repulsion, especially when the medium pH is far from neutral, and the bulk dielectric constant is low because of the presence of methanol. If Coulombic repulsion causes the unfolded chain to expand, then the expansion is expected to be variable with the charge content, meaning that the charge density is not highly dependent on pH. Proteins are thought to interact with alcohol predominantly through hydrophobic forces, and such preferential interactions are sensitive to local chain topography.11,14,28−30 As such, a chain expanded to a greater extent due to increased charge repulsion must somehow expose higher-affinity apolar surfaces with which methanol can interact. Many theoretical studies of solute interactions with macromolecules have been presented,11,27,31−35 but the interaction of uncharged solutes with a charged protein is not adequately known. 3.4. Charge-Dependent Molecular Expansion Is Related to the Magnitude of Δξ1. To emphasize protein expansion as the molecular basis for larger extents of 720

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apparent net charge contents the same in the folded and unfolded states. The kinetic traces (Figure 3a−c) show that

Figure 3. Stopped-flow kinetics for methanol-induced unfolding of cytochrome c at different pH values as indicated. The pH values of the initial solution and final unfolding solution in the stopped-flow experiment were held constant (see text). The initial protein solution was in aqueous medium, and the final methanol concentration in all experiments was 65%. (a) In the fluorescence-monitored kinetics, two rising exponentials characterized by the observed rate constants λ1 and λ2 describe unfolding at low pH. The slow unfolding phase changes to a refolding phase (fluorescence decreases) as the neutral pH region is approached. (b) In the neutral pH region, the slow phase appears to be a distinct refolding phase. Exponential fits to the data are rather poor in this region because of the nature of the kinetics. (c) In the basic pH region, the long-time unfolding phase appears again. (d) Variation of the observed rate constants, λ1 (blue) and λ2 (teal), with pH. The pH dependence of λ1 simulates the pH-dependent electrostatic free energy of the protein.

Figure 4. (a) Dependence of the effective number of water molecules preferentially excluded, |Δξw|, on the unfolding rate of cytochrome c. (b) Correlation between the hydrodynamic radius in aqueous solution, ⟨RH⟩, and |Δξw|. Values of each x,y coordinate are for a given pH.

the expectation that both Coulombic destabilization of the initial folded state relative to the transition state that lowers the activation enthalpy and the degree of water association of the folded state relative to the methanol-unfolded state increase with increased ionization of protein residues at acidic and basic pH values. 3.6. Correlation between ⟨RH⟩ and Δξ1. The loss of tertiary contacts and charge repulsions in acidic and basic pH regions is accompanied by molecular expansion (⟨RH⟩) and, indeed, also by larger protein−water interactions. The extent of water exclusion in methanol unfolding should then reflect the hydrodynamic dimensions of the initial protein state. This is shown in Figure 4b, where the correlation does not appear that strong (r2 = 0.65), perhaps because of measurement error. 3.7. Water Exclusion Results Are Independent of Protein Stability. The rationale for conducting pH-dependent experiments here was to generate differently stable cytochrome c molecules bearing different net charges so that preferential water interaction across the native → unfolded phase change could be examined with variable net charge contents. Although charge repulsions in pH ranges far from the neutral lead to partial denaturation and structural instability of the protein,43 the inferences regarding charge-dependent dynamics of protein-associated water are little affected, because the parameters of interest are measured in a charge-specific manner unlike in inter-pH experiments where the net charges for the initial and final states of the protein differ. In the equilibrium version of such experiments, it is the protein-associated waters in the initial and final states that matter, notwithstanding the thermodynamic stability of the protein, even though the

low-pH unfolding is associated with two unfolding phases, of which the slower one changes the sign of the amplitude and turns to a refolding phase when the neutral pH region is approached, but the two-phase unfolding behavior resurfaces at higher pH. The slow fluorescence-decreasing phase at intermediate pH (Figure 3b), where the native protein is most stable, could also be due to some nonrefolding event, the details of which will be examined in a later study. The pH dependence of the observed unfolding rate constant λ1 indicates increasing unfolding rates at acidic and basic pH values (Figure 3d). The observed dependence closely simulates the variation of the electrostatic free energy of cytochrome c across the pH scale.42 Although the value of λ1 does not change in the range of pH 6−8, the data can be taken to indicate faster unfolding with a growing number of preferentially excluded water molecules, Δξ1 (Figure 1b). This observation leads one to consider the dependence of λ1 on Δξ1. Indeed, a linear correlation (r2 = 0.83) can be observed (Figure 4a), suggesting similar pH dependences for the unfolding rate and end-state hydration. The correlation would appear to be consistent with 721

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The Journal of Physical Chemistry B number of waters involved is a consequence of structural unfolding due to Coulombic repulsion. The kinetic results, however, reproduce the expectation that unfolding accelerates with increasing thermodynamic instability when the net charge content increases (Figure 3a), and thus the unfolding rate, λ1, is correlated with the number of protein-associated water molecules that are excluded during unfolding (Figure 4a). 3.8. Miscellaneous Aspects of pH, pKa, and Component Interactions in the Water−Protein−Methanol System. In the context of methanol-induced changes in the structure and hydration of a protein, it is also important to consider the mutual effects of methanol and solution pH. Because of the higher pKa of methanol (>15), the pH of methanolic aqueous solutions is not considerably different from that of the aqueous phase alone.44 This is indeed the case even when the methanol content is as high as 75% by volume. However, competitions for intercomponent interactions arise under extremely basic conditions, where the solution pH approaches the pKa values of water, methanol, and protein backbone amides. As described earlier,10 all three components can now potentially interact with each other by H-bonding interactions. This mode of protein−alcohol interaction by direct H-bonding between backbone amides and the −OH group of the alcohol under highly basic conditions is distinct from the hydrophobic interaction that occurs between protein apolar surfaces and the −CH3 group of the alcohol. However, the hydrophobic interaction is the dominant mode irrespective of the solution pH.10 The transfer of the methanol −CH3 group from the solution to the apolar surfaces of the unfolded protein is accompanied by a large positive change of entropy (ΔS > 0), which outweighs any unfavorable enthalpy change. We conclude that methanol interacts with the protein at all working pH values through thermodynamically favorable hydrophobic forces.

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REFERENCES

(1) Meyer, E. Internal Water Molecules and H-Bonding in Biological Macromolecules: A Review of Structural Features with Functional Implications. Protein Sci. 1992, 1, 1543−1562. (2) Otting, G.; Liepinsh, E.; Wuthrich, K. Protein Hydration in Aqueous Solution. Science 1991, 254, 974−980. (3) Denisov, V. P.; Halle, B. Protein Hydration Dynamics on Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion. J. Mol. Biol. 1995, 245, 682−697. (4) Garcia, A. E.; Hummer, G. Water Penetration and Escape in Proteins. Proteins: Struct., Funct., Genet. 2000, 38, 261−272. (5) Kamal, J. K. A.; Zhao, L.; Zewail, A. H. Ultrafast Hydration Dynamics in Protein Unfolding: Human Serum Albumin. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13411−13416. (6) Svergun, D. I.; Richard, S.; Koch, M. H.; Sayers, Z.; Kuprin, S.; Zaccai, G. Protein Hydration in Solution: Experimental Observation by X-ray and Neutron Scattering. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2267−2272. (7) Levy, Y.; Onuchic, J. N. Water Mediation in Protein Folding and Molecular Recognition. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 389−415. (8) Gekko, K.; Timasheff, S. N. Thermodynamic and Kinetic Examination of Protein Stabilization by Glycerol. Biochemistry 1981, 20, 4677−4686. (9) Timasheff, S. N. Protein−Solvent Preferential Interactions, Protein Hydration, and the Modulation of Biochemical Reactions by Solvent Components. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9721− 9726. (10) Sashi, P.; Yasin, U. M.; Bhuyan, A. K. Unfolding Action of Alcohols on a Highly Negatively Charged State of Cytochrome c. Biochemistry 2012, 51, 3273−3283. (11) Hamsa Priya, M.; Merchant, S.; Asthagiri, D.; Paulaitis, M. E. Quasi-Chemical Theory of Cosolvent Hydrophobic Preferential Interactions. J. Phys. Chem. B 2012, 116, 6506−6513. (12) Lee, J. C.; Timasheff, S. N. Partial Specific Volumes and Interactions with Solvent Components of Proteins in Guanidine Hydrochloride. Biochemistry 1974, 13, 257−265. (13) Prakash, V.; Loucheux, C.; Scheufele, S.; Gorbunoff, M. J.; Timasheff, S. N. Interactions of Proteins with Solvent Components in 8 M Urea. Arch. Biochem. Biophys. 1981, 210, 455−464. (14) Timasheff, S. N. Protein−Solvent Interactions and Protein Conformation. Acc. Chem. Res. 1970, 3, 62−68. (15) Inoue, H.; Timasheff, S. N. Preferential and Absolute Interactions of Solvent Components with Proteins in Mixed Solvent Systems. Biopolymers 1972, 11, 737−743. (16) Scatchard, G. Physical Chemistry of Protein Solutions: Derivation of the Equations for the Osmotic Pressure. J. Am. Chem. Soc. 1946, 68, 2315−2319. (17) Kamatari, Y. O.; Konno, T.; Kataoka, M.; Akasaka, K. The Methanol-Induced Globular and Expanded Denatured States of Cytochrome c: A Study by CD, Fluorescence, NMR and SmallAngle X-ray Scattering. J. Mol. Biol. 1996, 259, 512−523. (18) Russell, B. S.; Melenkivitz, R.; Bren, K. L. NMR Investigation of Ferricytochrome c Unfolding: Detection of an Equilibrium Unfolding Intermediate and Residual Structure in the Denatured State. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8312−8317. (19) Davis, L. A.; Schejter, A.; Hess, G. P. Alkaline Isomerization of Oxidized Cytochrome c: Equilibrium and Kinetic Measurements. J. Biol. Chem. 1974, 249, 2624−2632. (20) Bhuyan, A. K. Off-Pathway Status for the Alkali Molten Globule of Horse Ferricytochrome c. Biochemistry 2010, 49, 7764−7773.

Because alcohols preferentially interact with protein hydrophobic surfaces exposed in the unfolded state, alcohol-induced equilibrium unfolding can be used to sense the effective number of water molecules expelled (Δξ1) during the native → unfolded transition. Both the unfolding rate and the degree of molecular expansion, characterized by ⟨RH⟩, are linearly correlated with Δξ1.

ASSOCIATED CONTENT

S Supporting Information *

Figure showing primary data for methanol-induced equilibrium unfolding of cytochrome c at different pH values. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

This research was supported by funds from Departments of Biotechnology (BRB/10/622/2008) and Science & Technology (4/1/2003-SF and SR/SO/BB-0075/2012) to A.K.B.

4. CONCLUSIONS

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 91-40-2313-4810. Notes

The authors declare no competing financial interest. A.K.B. generated ideas and designed research; P.S., U.M.Y., H.B., M.U.S., D.R., and A.K.B. performed research; P.S., U.M.Y., H.B., and A.K.B. analyzed data; and A.K.B. wrote the paper. 722

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(21) Chattopadhyay, K.; Mazumdar, S. Stabilization of Partially Folded States of Cytochrome c in Aqueous Surfactant: Effects of Ionic and Hydrophobic Interactions. Biochemistry 2003, 42, 14606−14613. (22) Latypov, R. F.; Cheng, H.; Roder, N. A.; Zhang, J.; Roder, H. Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c. J. Mol. Biol. 2006, 357, 1009−1025. (23) Bhuyan, A. K.; Udgaonkar, J. B. Folding of Horse Cytochrome c in the Reduced State. J. Mol. Biol. 2001, 312, 1135−1160. (24) Wyman, J., Jr. Linked Functions and Reciprocal Effects in Hemoglobin: A Second Look. Adv. Protein Chem. 1964, 19, 223−286. (25) Tanford, C. Extension of the Theory of Linked Functions to Incorporate the Effects of Protein Hydration. J. Mol. Biol. 1969, 39, 539−544. (26) Porras, S. P.; Sarmini, K.; Fanali, S.; Kenndler, E. Medium Effect (Transfer Activity Coefficient) of Methanol and Acetonitrile on βCyclodextrin/Benzoate Complexation in Capillary Zone Electrophoresis. Anal. Chem. 2003, 75, 1645−1651. (27) Manning, G. S. Limiting Laws and Counterion Condensation in Polyelectrolyte Solutions. I. Colligative Properties. J. Chem. Phys. 1969, 51, 924−933. (28) Tanford, C. Protein Denaturation. Adv. Protein Chem. 1968, 23, 121−282. (29) Thomas, P. D.; Dill, K. A. Local and Nonlocal Interactions in Globular Proteins and Mechanisms of Alcohol Denaturation. Protein Sci. 1993, 2, 2050−2065. (30) Arakawa, T.; Goddette, D. The Mechanism of Helical Transition of Proteins by Organic Solvents. Arch. Biochem. Biophys. 1985, 240, 21−32. (31) Mills, P.; Anderson, C. F.; Record, M. T. Grand Canonical Monte Carlo Calculations of Thermodynamic Coefficients for a Primitive Model of DNA−Salt Solutions. J. Phys. Chem. 1986, 90, 6541−6548. (32) Parsegian, V. A.; Rand, P. P.; Rau, D. C. Osmotic Stress, Crowding, Preferential Hydration, and Binding: A Comparison of Perspectives. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3987−3992. (33) Tang, K. E. S.; Bloomfield, V. Assessing Accumulated Solvent Near a Macromolecular Solute by Preferential Interaction Coefficients. Biophys. J. 2002, 82, 2876−2891. (34) Anderson, C. F.; Courtenay, E. S.; Record, M. T. Thermodynamic Expressions Relating Different Types of Preferential Interaction Coefficients in Solutions Containing Two Solute Components. J. Phys. Chem. B. 2002, 106, 418−433. (35) Shukla, D.; Shinde, C.; Trout, B. L. Molecular Computation of Preferential Interaction Coefficients of Proteins. J. Phys. Chem. B 2009, 113, 12546−12554. (36) Tanford, C. Protein Denaturation: Theoretical Model for the Mechanism of Denaturation. Adv. Protein Chem. 1970, 24, 1−95. (37) Yang, A. S.; Honig, B. Structural Origins of pH and Ionic Strength Effects on Protein Stability. J. Mol. Biol. 1994, 237, 602−614. (38) Alexov, E.; Gunner, M. Incorporating Protein Conformational Flexibility into Calculation of pH-Dependent Protein Properties. Biophys. J. 1997, 74, 2075−2093. (39) Shosheva, A.; Miteva, M.; Christova, P.; Atanasov, B. pHDependent Stability of Sperm Whale Myoglobin in Water−Guanidine Hydrochloride Solutions. Eur. Biophys. J. 2003, 31, 617−625. (40) Weinkam, P.; Pletneva, E. V.; Gray, H. B.; Winkler, J. R.; Wolynes, P. G. Electrostatic Effects on Funneled Landscapes and Structural Diversity in Denatured Protein Ensembles. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1796−1801. (41) Colonna-Cesari, F.; Sander, C. Excluded Volume Approximation to Protein−Solvent Interaction. The Solvent Contact Model. Biophys. J. 1990, 57, 1103−1107. (42) Hritsova, Sv.; Zhivkov, A.; Atanasov, B. Electrostatics of Horse Heart Cytochrome c and Montmorillonite Monolamellar Plate. Biotechnol. Biotechnol. Equip. 2009, 23, 568−571. (43) Goto, Y.; Nishikiori, S. Role of Electrostatic Repulsion in the Acidic Molten Globule of Cytochrome c. J. Mol. Biol. 1991, 222, 679− 686.

(44) Bosch, E.; Bou, P.; Allemann, H.; Roses, M. Retention of Ionizable Compounds on HPLC. pH Scale in Methanol−Water and the pK and pH Values of Buffers. Anal. Chem. 1996, 68, 3651−3657.

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dx.doi.org/10.1021/jp4111103 | J. Phys. Chem. B 2014, 118, 717−723