D Isotope Effects in Protein Thermal Denaturation: The Case of

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H/D Isotope Effects in Protein Thermal Denaturation: The Case of Bovine Serum Albumin Ling Fu,*,† Sandrine Villette,‡ Stephane Petoud,‡ Felix Fernandez-Alonso,§,|| and Marie-Louise Saboungi† †

Centre de Recherche sur la Matiere Divisee, UMR 6619-CNRS, Universite d’Orleans, 1b rue de la Ferollerie, 45071 Orleans Cedex 2, France Centre de Biophysique Moleculaire, UPR 4301-CNRS, rue Charle Sadron, 45071 Orleans Cedex 2, France § ISIS Facility, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, United Kingdom Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, United Kingdom

)

‡

ABSTRACT: The present work investigates the effects of H/D isotopic sub stitution on the structural and thermodynamic stability of bovine serum albumin (BSA) in aqueous solution over the temperature range of 5-90 °C. Using far-ultraviolet circular dichroism, we have compared protein unfolding pathways in H2O and D2O. Our results show that BSA possesses similar conformations in H2O and D2O at temperatures below 50 °C but follows different unfolding pathways at higher temperatures. The presence of D2O retards the occurrence of irreversible thermal denaturation in BSA, as evidenced by a higher onset temperature of 58 °C, in contrast to 50 °C in H2O. D2O exhibits a protective effect on the domain structure during the early stages of domain denaturation. Following incubation at 90 °C over a period of minutes, D2O causes a rapid aggregation of BSA molecules. This behavior is not observed in H2O solutions. Meanwhile, H/D substitution does not influence the reversible structural transformation of the protein in a significant manner. Partly renatured BSA in H2O and D2O undergoes very similar reversible structural transformations during a second heating cycle.

’ INTRODUCTION The functionality of a protein in biological processes is dependent upon its structure under physiological conditions. From a thermodynamic point of view, protein structure is determined by energetics. A protein molecule adopts the structure which minimizes the Gibbs free energy of the system. The challenge is to identify the important driving forces that dictate protein structure at the molecular level. Recent work indicates that protein structure is predominantly driven by a competition between intramolecular peptide hydrogen bonds and protein-solvent hydrogen bonds, each formed at the expense of the other (see ref 1 and references therein). Arguably, hydrophobic interactions between nonpolar side chains and solvent, previously regarded as the primary driving force behind protein folding,2 appear to have a secondary role. In either case, protein-solvent interactions are a central player in protein folding and stability. Although there exist other solvents, water is the universal solvent for life and can be found in every living organism. In a variety of experimental techniques for protein structure determination, it is standard procedure to dissolve the protein in D2O instead of H2O.3,4 In infrared spectroscopy, Raman scattering, and nuclear magnetic resonance, the signal from H2O may interfere considerably with those from the protein. In neutron scattering, D2O is used to prevent unwanted backgrounds arising from the incoherent scattering of protons.5 In applying the results of such measurements, it is often assumed that the protein r 2011 American Chemical Society

molecules have essentially the same structure in H2O and D2O, an approximation that should always be checked carefully. Due to smaller zero-point-energy motions, the hydrogen bonds in D2O are expected to be stronger than in H2O by 0.1-0.2 kcal mol-1,6 leading to an overall strengthening of hydrogen bonds. Although the energy difference associated with a single bond is small, the cumulative effects arising from deuterium isotopic substitution on macromolecules can be significant. In addition, quantum effects on the structure of water are amenable to study using neutron and X-ray scattering techniques. It has been shown that the additional quantum effects observed in H2O compared with D2O are approximately equivalent to a temperature rise of about 56 °C.7-9 Spectroscopic studies show that isotopic substitution in water from H to D shifts vibrational and librational modes to lower frequencies as a result of an increase in reduced mass.10 The strengthening effects following H/D substitution also apply to exchangeable N(H/D) 3 3 3 OC hydrogen bonds within the protein and hydrogen bonds between the protein and the solvent, and critically influence protein stability. Deuterium isotopic substitution effects on macromolecules are demonstrated by the fact that high concentrations of heavy water are toxic, and sometimes even lethal, to biological systems. Received: May 25, 2010 Revised: December 16, 2010 Published: February 3, 2011 1881

dx.doi.org/10.1021/jp104769v | J. Phys. Chem. B 2011, 115, 1881–1888

The Journal of Physical Chemistry B

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H/D isotopic substitution on protein stability has been studied in bulk thermodynamic measurements. Using differential scanning calorimetry (DSC), Efimova et al. showed that proteins tend to be more stable in D2O than in H2O, based on a comparison of the Gibbs energy of protein unfolding.11 They attributed this effect to an enhancement of the hydrophobicity in D2O causing the protein to reduce exposure of its hydrophobic side chains to the solvent. On the other hand, Makhatadze et al. has reported that RNase and lysozyme are less stable in D2O at room temperature on the basis of the Gibbs energy of unfolding.12 Both groups have also reported a higher protein denaturation temperature in D2O. The fluorescence study by Cioni and Strambini showed that D2O significantly increases protein rigidity, the effect being more marked at higher temperatures.13 However, neither DSC nor fluorescence measurements provide direct information on protein secondary structure, which is critical to biological function and activity. In the present work, we address this issue by following the thermal unfolding of bovine serum albumin (BSA) with far-UV circular dichroism (CD), a powerful technique sensitive to protein secondary structure.14-16 BSA (molecular weight 66 300 Da) is an abundant and widely studied protein. It has numerous biological functions and biochemical applications. The structure of native BSA is dominated by R-helices and contains no β-sheets.17 Its structure is composed of three homologous domains (I, II, and III) that unfold quite independently.

’ MATERIALS AND METHODS BSA was obtained from Sigma Chemical Company and used without further purification. H2O was distilled and deionized prior to the experimental runs, and the D2O isotopic purity was 99.9%. The protein was dissolved in a H2O/D2O 20 mM phosphate buffer at pH/D 7 followed by the addition of 150 mM NaCl. The pH meter reading of the D2O solutions was corrected according to the relationship pD = pH meter reading þ 0.4.18 The protein was incubated in D2O for at least 12 h to complete the H-D exchange process. Studies have shown that proteins incubated in D2O for several hours reach a level of exchange that is stable for at least 3 days.11 In the metastable configuration, all labile side chain H-atoms and part of the backbone H-atoms are exchanged, whereas the H-atoms involved in hydrogen bonding in R-helices and β-sheets are not exchangeable. The concentration of the protein was 0.2 mg/mL or 3 μM, as determined by UV absorption spectroscopy (A1mg/mL,1cm = 279nm 0.667). The CD experiments were carried out on a Jasco J-810 spectropolarimeter equipped with quartz Suprasil cuvettes (0.1 cm path length). The data were recorded over the temperature range of 5-90 °C, and the temperature in the cell was controlled with a circulating water-bath system. Each sample was heated from 5 to 90 °C at a heating rate of 1 °C/min, then immediately cooled down to 5 °C, and finally heated back to 90 °C at the same heating rate. The CD signal at 222 nm, widely used as an indicator of protein secondary structure,15 was recorded in 0.2 °C intervals, whereas the CD spectrum over the region of 190-250 nm was measured at 10 °C intervals with a bandwidth of 1 nm. Identical procedures were followed for both H2O and D2O solutions. In order to investigate BSA aggregation and irreversibility, we also tracked CD intensities at 222 nm for BSA incubated at high temperatures for 1 h and cooled back to 20 °C.

Figure 1. CD spectra of BSA in (a) H2O and (b) D2O at 10 (black), 70 (blue), and 90 °C (red) for the first (solid lines) and second heating (dashed lines). Units for ellipticity are mdeg cm2 dmol-1 res-1, where res denotes normalization of the CD data by the number of amino acid residues.

’ RESULTS AND DISCUSSION Thermal Transformation of the Secondary Structure. Figure 1 shows the CD spectra of BSA in H2O and D2O at three temperatures. At 10 °C, the spectra in both solutions show a double band characteristic of high R-helical content. As the temperature increases, the spectra evolve toward a shape characteristic of R-poor structures. Cooling back down to 10 °C does not reproduce the original spectra. The spectra measured during the second heating do not vary much with temperature and are similar to those taken at the higher temperatures during the first heating. Clearly, the first heating causes some irreversible transformation of the secondary structure of BSA, whereas subsequent heating does not cause further significant changes. A comparison of the CD spectra of the two solutions for the first heating is shown in Figure 2. As an indicator of the R/β ratio in the secondary structure, the temperature dependence of the CD signal at 222 nm, θ222, is shown in Figure 3. The curves for the first heating exhibit a sigmoidal profile with a slope that is steeper at high temperatures than at low temperatures. The denaturation process of BSA continues at 90 °C without saturation. BSA denaturation continues up to temperatures as high as 130 °C, as previously reported by Moriyama et al.19 As shown in Figure 3, the two first-heating transition curves are very similar to each other in the low-temperature region but start to deviate at around 50 °C, where θ222 increases significantly, indicating a major transformation of the secondary 1882

dx.doi.org/10.1021/jp104769v |J. Phys. Chem. B 2011, 115, 1881–1888


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Figure 2. CD spectra of BSA in H2O (red) and D2O (blue) at (a) 10, (b) 50, (c) 70, and (d) 90 °C during the first heating.

Figure 3. Temperature dependence of CD intensities at 222 nm for BSA in H2O (red) and D2O (blue) over the course of the first (solid line) and second (dashed line) heating.

structure. The substitution of D2O for H2O retards this transformation between 50 and 60 °C but accelerates it between 60 and 70 °C. Differences between the two curves are minimized at 70 °C and become evident again at higher temperatures. The irreversible transformation above 50 °C is associated with a disruption of the helical content and the appearance of β-sheet structures. We adopt the method of Greenfield and Fasman to derive the fractional R-helical content, fR, in BSA from the CD signal at 208 nm according to the relation fR = ([θ]208nm 4000)/(33000 - 4000).16 The result is shown in Figure 4. It is important to note that the disrupted helical structures do not show complete recovery upon cooling. As the temperature is raised to 90 °C during the second heating, the helicity slowly

decreases to values slightly lower than those measured at the end of the first heating. The β-sheet content is estimated using the fitting methods available in the online CDPro software package.20 The CONTINLL method determines that the β-sheet content of both solutions, 4% at room temperature, increases rapidly above 50 °C reaching a maximum of 24% at 90 °C. Upon cooling, the β-sheet content decreases to about 15%. The increase in the β-sheet content is delayed in the D2O solution between 50 and 70 °C and correlates with the decrease of R-helices shown in Figure 4. At 60 °C, the β content is estimated to be 13% in H2O, compared to 9% in D2O. According to the multistep denaturation mechanism proposed by Flora et al.,21 the structural transformation between 50 and 70 °C involves the irreversible unfolding of domain II. Further heating causes irreversible unfolding of domain I. On the basis of the above, our experimental results indicate that the presence of D2O slows down the structural transformation at the early stages of domain denaturation but promotes it at the later stages involving domain I. The fate of domain III during denaturation remains unclear, yet recent experiments by Moriyama et al.19 suggest that helical structures at the connecting segments between all three domains are also disrupted. At the early stages of protein unfolding, most H-atoms associated with the BSA backbone are not isotopically substituted when dissolved in D2O. The intramolecular backbone hydrogen bonds and the deuterium bonds in the solvent are energetically favored over the protein-solvent interactions due to a stronger affinity among D2O molecules. As denaturation proceeds, the backbone in the protein’s interior becomes increasingly exposed to the solvent. The hydrogen atoms forming the hydrogen bonds between the N-H and the CdO groups responsible for the stabilization of R-helices are then readily exchanged by deuterium. In this situation, the protective effect of the stronger deuterium 1883



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Ignoring a constant contribution from irreversibly disrupted BSA in the second heating, the observed CD signal, θ, can be written as the sum of two terms θ¼

1 K θN þ θU 1þK 1þK

ð1Þ

where θN and θU are the contributions to the CD signal of the structural units in states N and U, respectively, and K (= ([U])/([N])) is an equilibrium constant linked to the Gibbs free energy difference, U ΔU NG, via K = exp(-ΔNG/RT). With these considerations in mind, we can then write ΔUN G ¼ ΔUN HðTÞ-TΔUN SðTÞ

Figure 4. Temperature dependence of the fractional helical content of BSA in H2O (red) and D2O (blue) during the first (solid line) and second (dashed line) heating. The lines are guides to the eye.

where the enthalpy and the entropy satisfy the equations Z T ΔHðTÞ ¼ ΔHðTe Þ þ ΔUN Cp dT

ð2Þ

ð3Þ

Te

ΔSðTÞ ¼

Z T ΔHðTe Þ þ ΔUN Cp d ln T Te Te

ð4Þ

Here Te is the temperature where both states are equally probable. Assuming ΔU N C p to be temperature-independent, we obtain T T U U U -ΔN Cp ðTe Þ Te -TþT ln ΔN G ¼ ΔN HðTe Þ 1Te Te ð5Þ

Figure 5. CD signals at 222 nm of BSA in H2O (red) and D2O (blue) for the second heating. Here, θ222 of BSA in H2O is rescaled and shifted to overlay with θ222 of BSA in D2O.

bonds becomes weakened after the backbone protons are progressively replaced by deuteria. Reversible Thermal Transformation of the Secondary Structure. Our CD experiments indicate that, at the inception of the second heating, the partly denatured and aggregated BSA molecules contain two types of structural units, namely, those that were irreversibly disrupted during the first heating, denoted as species F, and those that had been reversibly modified during the first heating and restored to a native-like state after cooling, denoted as species R. The structural units of the R species are subjected to a reversible transformation again during the second heating. Tracking the changes of each structural unit is a complex exercise beyond the remit of this study and present capabilities of the CD technique. Our CD data, however, is consistent with the presence of an ensemble of reversible structural units, the R species, exhibiting a simple thermal equilibrium between two states, hereafter denoted as N and U. N represents the native and restored states of the R species, assumed in this treatment to be identical, and U corresponds to the partly unfolded state of the R species.

As shown in Figure 5, the 222 nm CD signal in H2O and D2O during the second heating is identical within a small multiplicative factor and a baseline shift, suggesting that the protein molecules are subjected to very similar reversible structural changes. The scaling factor and offset arise from the irreversibly denatured structural units. As such, they are not relevant in our analysis of the reversible thermal transformation between the N and U states. In thermodynamic terms, H/D substitution does not shift the N h U equilibrium, implying that the two solvents have an equal influence on the free energy of the N and U states. To reduce experimental uncertainties, we have averaged the two curves and fitted these data using nonlinear least-squares routines to eq 1, as shown in Figure 6. The best-fit parameters are Te=1.1102 °C, ΔH(Te) = 44 kJ mol-1, and ΔCp(Te) = 0.32 kJ K-1 mol-1. The values of ΔU NG(T) calculated from eq 5 using our best-fit parameters are shown in Figure 7. The parabolic stability profile predicts a lowtemperature equilibrium near -134 °C, close to that hypothesized for the glass transition of water.22 Unfortunately, such a low temperature cannot be accessed without the formation of ice, unless other agents are added to the solvent. Correlations between the First and Second Thermal Transition Curves. We have classified the secondary structure units of BSA into two species, R and F. The R species undergoes a reversible transformation that can be restored on cooling, whereas the transformation of the F species is irreversible. During the first heating, the CD signals for BSA can therefore be written as a linear combination of these two species: θ1 ¼ fR ðTÞθR ðTÞ þ fF ðTÞθF ðTÞ

ð6Þ

where θR(T) and θF(T) are the average CD signals from the R and F species and fR(T) and fF(T) represent the fractions that are 1884



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Figure 6. Average CD intensities at 222 nm for BSA in H2O and D2O during the second heating (dots) and associated least-squares fit (line). See the main text for further details.

Figure 7. Temperature dependence of the Gibbs free energy obtained from the fit shown in Figure 6. See the main text for further details.

transformed at a given temperature T reversibly and irreversibly, respectively. At the end of the first heating, fF(T) and θF(T) reach their maximum values, fF,max and θF,max. The conformational changes during cooling and second heating are found to be essentially reversible, so fF and θF remain at these values. Thus, during the second heating, the CD signal becomes θ2 ¼ ð1-fF, max ÞθR ðTÞ þ fF, max θF, max

ð7Þ

From eqs 6 and 7, we obtain θ2 ¼

1-fF, max fF ðTÞ θ1 þ fF, max θF, max -ð1-fF, max Þ θF ðTÞ ð8Þ fR ðTÞ fR ðTÞ

For temperatures lower than the onset temperature for irreversible changes, we may assume that fF ≈ 0 and fR ≈ 1. On the basis of these considerations, the two heating transition curves should then display a temperature-independent linear correlation θ2 ¼ ð1-fF, max Þθ1 þ fF, max θF, max

ð9Þ

Figure 8 shows plots of θ2 against θ1 for the two solutions. These data obey a linear relationship at low temperatures, in line with the predictions of eq 9. A linear regression analysis of these data shows that fF,max is 0.56 for H2O, and 0.63 for D2O. The curves deviate from linearity at about 50 °C for H2O and 58 °C for D2O, representing the threshold temperatures at which structural changes become irreversible. The denaturation temperature determined in this manner is in excellent agreement with the observation that the denaturation of BSA becomes irreversible only at temperatures higher than 50 °C.23,24 The presence of D2O raises the temperature threshold for irreversible denaturation and effectively stabilizes the BSA molecule between 50 and 58 °C. This observation is also consistent with previous experimental results.11-13 We find that the parameters fF(T) and fR(T) are slightly dependent on heating rate. An increase in heating rate from 1 to 2 °C/min results in slight changes in these parameters, particularly above the critical temperature where they tend to increase in a slower fashion. The reversible process can then be regarded

Figure 8. Correlation between CD intensities at 222 nm during the second (θ2) and first heating (θ1): (a) BSA in H2O and (b) BSA in D2O. The dashed lines are linear fits below 45 °C.

instantaneous compared to the time scales of our measurements, while the irreversible one takes place over a period of a few seconds. CD Results after High-Temperature Incubation. Figure 9 shows the time evolution of the CD signal θ222 for BSA in H2O and D2O after rapid (∼3 min) temperature changes from 20 °C to higher temperatures (60, 70, 80, and 90 °C), and back down to 20 °C after incubation for 1 h. Consistent with the results already 1885



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Figure 9. BSA CD signals, θ222, after stepwise temperature changes from 20 to (a) 60, (b) 70, (c) 80, and (d) 90 °C, and back to 20 °C. Changes during the heating and cooling periods (∼3 min) are not shown. Red: BSA in H2O. Blue: BSA in D2O.

shown, we observe an immediate increase in the CD signal on heating, with a lower negative ellipticity for H2O than for D2O at 60, 70, and 80 °C. Upon incubation at these temperatures, the CD signal θ222 continues to increase, albeit at a much slower rate. This gradual change was also observed by Vaiana et al. in parallel circular dichroism and dynamic light scattering experiments and was attributed to aggregation processes.25 Upon cooling to 20 °C, the secondary structure disrupted at the higher temperatures is partially restored. For the solution incubated at 60 °C, near the critical temperature for the irreversible transition, the restored BSA structure in H2O shows a smaller negative ellipticity than in D2O, whereas CD signals incubated at 70 and 80 °C returned to approximately the same value upon cooling in both H2O and D2O. For the D2O solution incubated at 90 °C, the CD signals change rapidly with time. We surmise that this process correlates with the irreversible formation at high temperature of high-order oligomers, possibly involving disulfide bonds. These bonds are likely to become increasingly exposed during the late stages of domain I unfolding, as this domain contains one free Cys residue. After cooling down to 20 °C, the CD signals retain their hightemperature values, indicating that the disruption of the secondary structures during the incubation at 90 °C is essentially irreversible. Assuming first-order kinetics for the aggregation process, the BSA fraction leading to oligomer formation at a

Figure 10. BSA CD signals at 222 nm after incubation at 90 °C in D2O. The curve is a fit to a first-order irreversible process with k = 6.7 10-4 s-1. The experimental conditions are the same as those in Figure 9d.

given time t is given by f = 1 - e-kt. From our CD data, we obtain a first-order rate constant of k = 6.7 10-4 s-1, as shown in Figure 10. 1886


The Journal of Physical Chemistry B The temperature dependence of the rate constant k also follows Arrhenius law, namely, k = A e-Ea/RT. For the present case, Ea represents the energy required for aggregation. For BSA dissolved in D2O, the fact that a change in CD intensities during the incubation period is evident at 90 °C, but much less so at 60, 70, and 80 °C, implies a critical temperature Tc (= Ea/R) between 80 and 90 °C, corresponding to an activation energy of about 3 kJ mol-1 for aggregation processes. In contrast, the CD signal for H2O remains flat at 90 °C, implying a critical temperature above 90 °C. The finding that the presence of D2O promotes aggregation is in accord with previous observations on other proteins.26,27 It is possible that continued exposure to UV radiation during our CD experiments may also contribute to the irreversible aggregation of BSA molecules at high temperatures. Given the large differences observed for BSA in H2O and D2O, we estimate that these effects are small as light absorption in the UV is largely insensitive to isotopic substitution. Moreover, UV exposure was the same for all samples investigated, yet distinct differences have been observed in CD response as a function of deuteration, temperature, and thermal history.

’ CONCLUSION Protein unfolding has been traditionally interpreted within the framework of a two-step Lumry-Eyring model involving a reversible transition from the native to a partly unfolded state K

N sF Rs U followed by an irreversible transition from the partly unfolded state to a final state28,29 k

Uf s F Our CD experiments show that the irreversible and reversible transitions of BSA occur simultaneously above the onset temperature of the irreversible transition. To address this additional complication, we have developed a novel method to determine the onset temperature via a linear correlation of the first- and second-heating transition curves at low temperatures. The onset temperature for BSA irreversible denaturation is 50 °C in H2O, and it is pushed up to 58 °C in D2O. In contrast, the reversible transformation is not affected appreciably upon isotopic substitution. Our experimental results indicate that BSA follows different unfolding pathways in H2O and in D2O. It therefore challenges in a fundamental way H-D substitution strategies widely used in a whole range of experimental techniques, as well as places important constraints on the conditions of its general applicability to explore protein structure and dynamics. Studies on the differences in protein structure in H2O and in D2O also provide important insights into the role of hydrogen bonds in protein denaturation. At the molecular level, isotope effects can be traced back to a decrease in zero-point-energy motions upon deuterium substitution, leading to an overall strengthening of both inter- and intramolecular hydrogen bonds. The interplay between these is a complex and multifaceted one. As a consequence, both the thermodynamic and kinetic behavior of the deuterated system may be significantly different from that displayed by its protonated counterpart.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]

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’ ACKNOWLEDGMENT The authors thank David Price and Michel Koch for a critical reading of the manuscript and Simon Billinge for helpful discussions. F.F.-A. gratefully acknowledges the hospitality and financial support from the CNRS Centre de Recherche sur la Matiere Divisee and the Universite d’Orleans. This study was supported by Agence Nationale de la Recherche (BIOSTAB program). ’ REFERENCES (1) Bolen, D. W.; Rose, G. D. Structure and Energetics of the Hydrogen-bonded Backbone in Protein Folding. Annu. Rev. Biochem. 2008, 77, 339–362. (2) Nemethy, G.; Sheraga, H. A. Structure of Water and Hydrophobic Bonding in Proteins. IV. the Thermodynamic Properties of Liquid Deuterium Oxide. J. Chem. Phys. 1964, 41 (3), 680–689. (3) Hedoux, A.; Ionov, R.; Willart, J.-F.; Lerbret, A.; Affouard, F.; Guinet, Y.; Descamps, M.; Prevost, D.; Paccou, L.; Danede, F. Evidence of a Two-stage Thermal Denaturation Process in Lysozyme: A Raman Scattering and Differential Scanning Calorimetry Investigation. J. Chem. Phys. 2006, 124, 014703. (4) Hedoux, A.; Willart, J.-F.; Paccou, L.; Guinet, Y.; Affouard, F.; Lerbret, A.; Descamps, M. Thermostabilization Mechanism of Bovine Serum Albumin by Trehalose. J. Phys. Chem. B 2009, 113 (17), 6119– 6126. (5) Saboungi, M.-L.; Price, D. L.; Mao, G.; Fernandez-Perea, R.; Borodin, O.; Smith, G. D.; Armand, M.; Howells, W. S. Coherent Neutron Scattering from PEO and a PEO-based Polymer Electrolyte. Solid State Ionics 2002, 147, 225–236. (6) Scheiner, S.; Cuma, M. Relative Stability of Hydrogen and Deuterium Bonds. J. Am. Chem. Soc. 1996, 118 (6), 1511–1521. (7) Badyal, Y. S.; Saboungi, M.-L.; Price, D. L.; Shastri, S. D.; Haeffner, D. R.; Soper, A. K. Electron Distribution in Water. J. Chem. Phys. 2000, 112 (21), 9206–9208. (8) Badyal, Y. S.; Price, D. L.; Saboungi, M.-L.; Haeffner, D. R.; Shastri, S. D. Quantum Effects on the Structure of Water at Constant Temperature and Constant Atomic Density. J. Chem. Phys. 2002, 116 (24), 10833–10837. (9) Fu, L.; Bienenstock, A.; Brennan, S. X-ray Study of the Structure of Liquid Water. J. Chem. Phys. 2009, 131, 234702. (10) Max, J.-J.; Chapados, C. Isotope Effects in Liquid Water by Infrared Spectroscopy. J. Chem. Phys. 2002, 116 (11), 4626–4642. (11) Efimova, Y. M.; Haemers, S.; Wierczinski, B.; Norde, W.; van Well, A. A. Stability of Globular Proteins in H2O and D2O. Biopolymers 2006, 55 (3), 264–273. (12) Makhatadze, G. I.; Clore, G. M.; Gronenborn, A. M. Solvent Isotope Effect and Protein Stability. Nat. Struct. Biol. 1995, 2 (10), 852–855. (13) Cioni, P.; Strambini, G. B. Effect of Heavy Water on Protein Flexibility. Biophys. J. 2002, 82, 3246–3253. (14) Johnson, W. C. Analyzing Protein Circular Dichroism Spectra for Accurate Secondary Structures. Proteins: Struct., Funct., Genet. 1999, 35, 307–312. (15) Chen, R. F.; Yang, J. T.; Chau, K. H. Determination of the Helix and β Forms of Proteins in Aqueous Solution by Circular Dichroism. Biochemistry 1974, 13 (16), 3350–3359. (16) Greenfield, N. J.; Fasman, G. D. Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation. Biochemistry 1969, 8 (10), 4108–4116. (17) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Crystal Structure of Human Serum Albumin at 2.5Å Resolution. Protein Eng. 1999, 12 (6), 439–446. (18) Schowen, B. K.; Schowen, R. L. Solvent Isotope Effects of Enzyme Systems. Methods Enzymol. 1982, 87, 551–606. (19) Moriyama, Y.; Watanabe, E.; Kobayashi, K.; Harano, H.; Inui, E.; Takeda, K. Secondary Structural Change of Bovine Serum Albumin in Thermal Denaturation up to 130°C and Protective Effect of Sodium 1887



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Dodecyl Sulfate on the Change. J. Phys. Chem. B 2008, 112, 16585– 16589. (20) Sreerama, N.; Woody, R. W. Estimation of Protein Secondary Structure from CD Spectra: Comparison of CONTIN, SELTON and CDSSTR Methods with an Expanded Reference Set. Anal. Biochem. 2000, 287, 252–260. (21) Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Bright, F. V. Unfolding of Acrylodan-labeled Human Serum Albumin Probed by Steady-state and Time-resolved Flouorescence Methods. Biophys. J. 1998, 75 (2), 1084–1096. (22) Velikov, V.; Borick, S.; Angell, C. A. The Glass Transition of Water, Based on Hyperquenching Experiments. Science 2001, 294 (5550), 2335–2338. (23) Anderle, G.; Mendelsohn, R. Thermal Denaturation of Globular Proteins—Fourier-Transform Infrared Studies of the Amide-III Spectral Region. Biophys. J. 1987, 52 (1), 69–74. (24) Lin, V.; Koenig, J. L. Raman Studies of Bovine Serum Albumin. Biopolymers 1976, 15 (1), 203–218. (25) Vaiana, S. M.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Irreversible Formation of Intermediate BSA Oligomers Requires and Induces Conformational Change. Proteins: Struct., Funct., Bioinf. 2004, 55 (4), 1053–1062. (26) Baghurst, P. A.; Nichol, L. W.; Sawyer, W. H. The Effect of D2O on the Association of Lactoglobulin A. J. Biol. Chem. 1972, 247 (10), 3199–3204. (27) Chakrabati, G.; Kim, S.; Gupta, M. L.; Barton, J. S.; Himes, R. H. Stabilization of Tubulin by Deuterium Oxide. Biochemistry 1999, 38 (10), 3067–3072. (28) Lumry, R.; Eyring, H. Conformation Changes of Proteins. J. Phys. Chem. 1954, 58 (2), 110–120. (29) Sanchez-Ruiz, J. M. Theoretical Analysis of Lumry-Eyring Models in Differential Scanning Calorimetry. Biophys. J. 1992, 61, 921–935.

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D Isotope Effects in Protein Thermal Denaturation: The Case of

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