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Back | Show only these items: | Solvation properties and stability of ribonuclease A in normal and deuterated water studied by dielectric relaxation and differential scanning/pressure perturbation calorimetry

1
Solvation properties and stability of ribonuclease A in normal and deuterated water studied by dielectric relaxation and differential scanning/pressure perturbation calorimetry

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

A change in solvent can have dramatic effects on the physico-chemical properties of a protein and its stability.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

In this paper we demonstrate by a study of solutions of the enzyme ribonuclease A (RNase A) in normal water (H₂O) and deuterated water (D₂O), to what extend a solvent isotopic substitution affects the structural and dynamic properties of a protein and its stability.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

Differential scanning calorimetry (DSC) indicates a shift of the transition temperature from the native to the unfolded state from about 62 °C in H₂O to 66 °C in D₂O. Pressure perturbation calorimetry (PPC), a relatively new and efficient technique, is used to study the volumetric properties of RNase A in its native and unfolded state.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met1

In PPC, the coefficient of thermal expansion of the partial volume of the protein, α, is deduced from the heat consumed or produced after small isothermal pressure jumps (±5 bar).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

α and its temperature coefficient, dα/dT, strongly depend on the interaction of the protein with the solvent at the protein–solvent interface.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

Both quantities are markedly affected by H₂O/D₂O substitution.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

Dielectric spectroscopy in the MHz and GHz regime is used to characterize the H₂O/D₂O isotope effect upon the tumbling time and dipole moment of the protein.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

The analysis of the isotope effects gives evidence for a decrease of the dipole moment and hydrodynamic radius of the protein in D₂O. An intriguing result is that the observed changes in thermodynamic properties reflect not only a stronger and more compact hydration in D₂O, but also an increase in protein compactness.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

A similar result is obtained from dielectric relaxation experiments on another small globular protein, ubiquitin.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

Introduction

Many protein functions are known to depend drastically on the structure and dynamics of the solvent.^1–5

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

For example, Fenimore et al⁵. have distinguished between “slaved” and “non-slaved” protein processes, depending on whether they are coupled to the solvent fluctuations or not.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

Because the coupling of the protein to the surrounding solvent bath is controlled by the properties of the protein–solvent interface, the characterization of protein hydration is essential for understanding these phenomena.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

This does not only require an understanding of the solvent effects upon the properties of the protein, but, vice versa, also of the effect of the protein, for instance the topology of the protein surface and its interfacial dynamics, on the solvent.^6–8

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

The present paper aims at contributing to a more detailed understanding of protein hydration by investigating effects of H₂O–D₂O substitution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

Isotopic substitution is used in many experiments on biomolecular systems to exploit properties of the deuterium nucleus not shown by protons, or to generate solute/solvent contrast.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

For example, the properties of the deuterium nucleus render D₂O to be the solvent of choice in neutron scattering studies of protein and hydration^3,9 and in NMR studies of biomolecular and hydration dynamics.^10–13

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

Deuterium is also used as a tracer, for instance, in kinetic studies of enzymatic reactions.¹⁴

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

Thereby, it is commonly assumed that the biomolecular interactions are not modified by the solvent isotopic substitution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

In contrast, we focus here on the structural and dynamical changes associated with protein–water interactions in H₂O and D₂O, respectively, and their consequences for the thermodynamic properties of the system.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

We consider the enzyme ribonuclease A (RNase A, 124 amino acids, molecular mass M = 13.7 kDa) as a prototypical protein used in many studies of protein folding.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj2

RNase A is a single-domain pancreatic enzyme protein which catalyses the cleavage of single-stranded RNA.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

Its crystal¹⁵ and solution¹⁶ structure comprise three helices and a large β-sheet region.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

It is known since a long time that H₂O–D₂O substitution increases the thermal unfolding temperature of RNase A.¹⁷

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

Such temperature shifts are found with other proteins as well.^18–21

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

Some additional measurements were also performed, for comparison, for the small globular protein ubiquitin (76 amino acids, M = 8565 Da).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

At the molecular level, H₂O–D₂O isotope effects result from the fact that deuterium bonds in water are stronger than hydrogen bonds, because the larger mass of the deuteron lowers the zero-point vibrational energy of the intermolecular modes.^22,23

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

The resulting increase in the solvent structure of D₂O and in D₂O–D₂O affinity reveals itself in distinct isotope effects on the thermodynamic and dynamical properties of pure water and aqueous solutions of simple solutes.²⁴

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

The rationale is that the increase in solvent structure causes a stronger solvation of hydrophilic and a less efficient solvation of hydrophobic species.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

In protein solutions, such effects might cause polypeptides to reduce their solvent exposure by adopting a more compact shape or by associating into larger aggregates.^19,25–28

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

This conjecture does not only explain the observed temperature shifts of unfolding transitions, but also the promotion of protein aggregation in D₂O.^27,28

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

However, such an interpretation is not unambiguous, and by a detailed analysis of entropy and enthalpy contributions, some authors have come to the opposite conclusion that the native protein should be destabilized by D₂O.^20,21

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

Important information on properties of the protein–water interface can also be deduced from volumetric properties.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

For example, it has been shown that the apparent thermal expansion coefficient, α, of the protein and its temperature coefficient, dα/dT, are drastically influenced by protein–solvent interactions.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

By a rather new and sensitive technique called pressure perturbation calorimetry (PPC), α(T) can now be measured with very high precision.^29–31

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met1

In PPC, the coefficient of thermal expansion of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met1

Using this technique, we have recently studied the solvation properties RNase A in its native and unfolded state in the presence of several chaotropic and kosmotropic co-solvents.³⁰

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

In the course of these studies we have found that the substitution of H₂O by D₂O has drastic effects on the volumetric properties, indicating a stabilization of the native form in D₂O. In the present work, we reconsider these isotope effects in more detail, and probe local isotope effects at the protein–water surface by dielectric relaxation experiments.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

Dielectric relaxation in the MHz region is a standard technique for studying the structure and dynamics of protein solutions.^32,33

Type: Method | Advantage: None | Novelty: None | ConceptID: Met2

Recent extensions over a broader frequency band up to the GHz regime^34,35 and data evaluation assisted by molecular dynamics (MD) simulations^36,37 considerably increase its power.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met2

Methods

Samples

Solutions of bovine pancreatic RNase A (Sigma Chemical Co.) were prepared with 10 mM Na₂HPO₄ buffer.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

The experiments in H₂O were performed at pH = 5.5, at which DSC traces indicated the highest stability of the native form.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

Solutions in D₂O (Aldrich, D-content > 99.9 atom-%) were adjusted to the same proton activity as in H₂O. This implies that the value of pD, e.g. recorded by a conventional glass electrode was 5.9 (i.e. pH + 0.4).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

Lyophilized and essentially salt-free ubiquitin from bovine red blood cells (Fluka, protein content > 90%) was dissolved in buffer-free H₂O and D₂O solutions.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

DSC and PPC experiments

The thermal unfolding of RNase A in H₂O and D₂O was measured by means of a high precision VP DSC micro-calorimeter (MicroCal, Northamption, MA, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

The same instrument, supplemented by the MicroCal PPC accessory, was used in the PPC experiments.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

Experimental details can be found in ^{refs. 29 and 30}.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

As the PPC technique is comparatively new, a brief description is given here.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

PPC measures the heat consumed or released by a sample after small isothermal pressure jumps.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

In the differential PPC experiment two cells of equal volume (here 0.5 mL), containing the protein solution and the buffer, are subject to the same pressure jump.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

In a decompression step, a pressure of 5 bar (here 5 bar by using nitrogen) is applied and is then released to ambient pressure.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

After equilibration, the gas is used to initiate a compression step.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

During the pressure jumps, constant temperature is achieved by active compensation of the heat changes.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

Integration of the supplied power yields the heat released or consumed.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

The heat peaks in the compression and decompression steps should be equal in value, but are of opposite sign.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

Thermodynamics relates the pressure coefficient (∂Q_rev/∂p)_T of the heat Q_rev exchanged in a reversible process to the coefficient of thermal expansion, α = (1/V)(∂V/∂T)_p, of the sample volume.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

For a sufficiently dilute solution containing m_S grams of solute and m₀ grams of solvent, the volume is given by V = m₀V₀ + m_SV̄_S, where V₀ is the specific volume of the solvent and V̄_S the partial specific volume of the solute.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

One then finds (∂Q_rev/∂p)_T = −TVα = −T[m₀V₀α₀ + m_SV̄_S_S].α₀ = (1/V₀)(∂V₀/∂T)_p and _S = (1/V̄_S)(∂V̄_S/∂T)_p are the coefficients of thermal expansion associated with the solvent volume and the solute partial volume, respectively.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

In a differential PPC experiment, the volume occupied by the solute in the sample cell, m_SV̄_S, is replaced by the same volume of solvent in the reference cell.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

Within small pressure intervals, the pressure dependence of V and α can be neglected, and eqn. (1) can be integrated to yield the working equation ΔQ_rev = −T[m_SV̄_S_S − m_SV̄_Sα₀]Δp Then, _S can be determined, if α₀ is known.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

In practice, it is a good approximation, to replace the partial specific quantities _S and V̄_S by the corresponding apparent quantities, in the following denoted by α and V, respectively.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

Thus, all volumetric properties refer to apparent molar volumes and apparent expansibilities of the protein.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

Dielectric relaxation

Dielectric spectroscopy monitors the real part (dielectric dispersion ε′(ω)) and imaginary part (dielectric loss ε″(ω)) of the complex dielectric permittivity of a sample as a function of the angular frequency ω = 2πν.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

Accounting processes in the optical and vibrational regime by an effective limiting value ε_∞ of the real part, the complex dielectric permittivity is given by^32,33,38ε*(ω) = ε′(ω) − iε″(ω) = ε_∞ + Δε′(ω) − iΔε″(ω) + σ/iε₀ω (i² = −1).ε″(ω) shows a low-frequency divergence which depends on the static (dc) conductivity, σ.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

The latter contribution can be determined at low frequencies, where only the σ/iε₀ω term survives.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

ε₀ is the permittivity of the vacuum.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

The relaxational contributions, Δε′ and Δε″(ω), depend on the same set of microscopic parameters.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

In the first set of experiments, we used the network analyzer 8712 ES from Agilent Technology (former Hewlett Packard) in combination with a coaxial line terminating in a home-made sample cell described by Kaatze and coworkers³⁹ to measure ε*(ω) in the range from ν = 1 MHz to ν = 1.3 GHz.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

In the second series, the network analyzer HP 8720 (Hewlett Packard) was used at 200 MHz ≤ ν ≤ 20 GHz in combination with the commercially available probe HP 85070B.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

Results and data evaluation

DSC results

Fig. 1 shows the background-corrected DSC traces of the buffered RNase A solution in H₂O and D₂O at a protein concentration of 0.5 wt.%.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

In H₂O, the protein begins to unfold at about 55 °C, and the temperature of the unfolding midpoint is T_m = (62.0 ± 0.2) °C.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

Table 1 summarizes the resulting thermodynamic data.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

The enthalpy change, obtained by integration over the DSC peak, is ΔH = (435 ± 4) kJ mol⁻¹.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

The increase in the apparent molar heat capacity from the native to the unfolded state, ΔC_p = (5.2 ± 0.2) kJ mol⁻¹ K⁻¹, is typical for proteins in H₂O. Substitution by D₂O shifts T_m to (66.2 ± 0.2) °C.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

The enthalpies of unfolding in H₂O by D₂O are almost the same, but ΔC_p in D₂O is only about half of that in H₂O.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

PPC results

Fig. 1 shows that the maxima in C_p at the unfolding transition correspond to minima in the temperature dependence of the apparent thermal expansion coefficient, α(T).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

T_m values extracted from the PPC curves agree within experimental error with those determined from the DSC traces.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

Volumetric data derived from the PPC curves are summarized in Table 1.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

In the native regime the PPC curves in both H₂O and D₂O show two major features: First, the apparent thermal expansion coefficient, α, of the protein is high, for example α₁₀ = 0.76 × 10⁻³ K⁻¹ at 10 °C in H₂O. Second, there is a steep slope in the temperature dependence of α(T).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

In H₂O between 10 and 40 °C α decreases almost linearly with a slope dα/dT = −4.3 × 10⁻⁶ K⁻².

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

The curve of α(T) in D₂O lies above that of H₂O, and α is more steeply decreasing with increasing temperature.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

Quantitatively, α in D₂O is by about 17% higher than in H₂O, and the negative slope, dα/dT, is enhanced by 46% (Table 1).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

In both H₂O and D₂O, α increases upon unfolding.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

The increase, Δα = 1.6 × 10⁻⁴ K⁻¹, is typical for proteins.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

The relative volume change upon unfolding can be calculated by integration of the α(T) transition curve after baseline subtraction.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

In H₂O, we obtain ΔV/V = −2.7 × 10⁻³.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

Based on the partial specific volume of RNase A (0.704 mL g⁻¹), the absolute volume change is ΔV = −26 mL mol⁻¹, which amounts to 0.27% of the total protein volume.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

In D₂O, the volume change is 40% below the value found for H₂O (Table 1).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

Dielectric relaxation data

We have determined dielectric relaxation spectra of solutions of RNase A (1.5 wt.%) and ubiquitin (2.5 wt.%) in H₂O and D₂O at 25 °C.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

The RNase A concentration was chosen as a compromise between the desire to reproduce the conditions of the PPC experiments and the need for sufficient amplitudes of the protein peaks in the spectra.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

Fig. 2 shows as an example the real and conductivity-corrected imaginary parts of the permittivity of RNase A in H₂O. The spectra exhibit a multi-modal structure with diffusive, so-called “Debye behavior” of each dispersion step, characterized by amplitudes S_j and relaxation times τ_j:³⁸ For an accurate representation of the spectrum of RNase A, four terms were needed.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

Numbering the dispersions from low to high frequencies, processes 1 and 4 are dominant and cause the apparent bimodal shape of the spectrum shown in Fig. 2.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

The low-frequency dispersion, often termed “β-relaxation”, results from protein tumbling, the high-frequency dispersion 4 reflects bulk water reorientation.^32–37

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

The interpretation of two further processes (2 and 3) at intermediate frequencies is subject to some debate, but probably they are related to protein–water interactions.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

At the low concentrations applied here, their amplitudes were too weak to be able to extract meaningful parameters.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

Processes 2 and 3 will not be considered further.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

In the case of ubiquitin, it made only sense to fit a three-term expression, as the difference in frequency of dispersions 2 and 3 was small.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

The fit parameters are summarized in Table 2.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

Discussion

A scenario for the volumetric behaviour

Before considering changes in the volumetric behavior induced by H₂O–D₂O substitution, we discuss a simple scenario, in which the partial specific volume of a protein may be decomposed into three contributions:^40–42V ≈ V_intr + δV_hydr + V_therm.The intrinsic volume, V_intr, of the protein results from the van der Waals volume of the atoms plus the volume of water-inaccessible voids in its interior.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

100

The hydrational or interaction term, δV_hydr, describes the solvent volume changes associated with the hydration of the solvent-accessible hydrophobic, polar or charged protein atomic groups.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

101

The thermal volume, V_therm, the volume of the void space surrounding the solute molecule, arises from mutual thermally induced vibrations and reorientations of the solute and solvent.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

102

Scaled particle theory, by employing statistical mechanical and geometrical arguments to describe the dissolution of a solute, allows one to evaluate the intrinsic and thermal contributions (^{ref. 42} and references therein).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

103

The thermal volume contribution has been found to be slightly larger in D₂O than in H₂O, respectively.⁴²

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

104

The sum of the intrinsic (geometrical) volume and the thermal volume thus represents the partial molar volume of the cavity enclosing the solute.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

105

In eqn. (5), a minor term taking into account the coefficient of isothermal compressibility of the solvent has been neglected.⁴⁰

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

106

Certainly, as there is no rigorous way of disentangling the partial molar volume of a protein into its components here, other dissections of V may be conceivable.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

107

Owing to the qualitative discussion of the various contributions, this does not affect the conclusions drawn, however.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

108

From the measured partial specific volume and simple models for V_intr and V_therm, a rough estimate of δV_hydr can be given for the native state.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

109

Such an analysis, conducted for RNase A in H₂O in^{ref. 30}, yields as a highly negative value for δV_hydr, about δV_hydr = −0.2 cm³ g⁻¹.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

110

A negative value implies a smaller partial molar volume, i.e. a higher density, of water at the protein surface compared to bulk water.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

111

Such a higher density is evidenced by combined neutron and X-ray scattering experiments.⁴³

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

112

Its origin in terms of the properties and topology of the protein–water surface has recently been addressed by MD simulations.⁸

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

113

Eqn. (5) implies that a similar dissection may hold for the temperature derivatives of the partial specific volume, i.e. the apparent thermal expansion coefficient, α, and its temperature coefficient, dα/dT.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

114

Only δV_hydr and V_therm contribute significantly, to α and dα/dT, because the intrinsic volume of the native protein does not depend markedly on temperature.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

115

In fact, the thermal expansivity of the protein interior has been measured over a limited temperature range, and the changes observed are rather small.^44–48

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

116

The thermal volume is expected to increase with temperature, giving a positive contribution to α.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

117

As pointed out by Lin et al.,²⁹α and, in particular, dα/dT are primarily controlled by the hydrational contributions, suggesting that dα/dT is a direct measure of the effect of solvation upon volumetric properties of proteins.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res12

118

In fact, the hydrational contribution to the thermal expansibility is known to depend drastically on the nature of the protein–water interface.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

119

Hydrophilic groups in contact with water show the characteristic pattern of structure-breakers with large positive values of α, which decrease drastically with increasing temperature.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

120

The rationale is that the hydrophilic groups bind more adjacent water at low temperatures, which at higher temperatures are released by thermal agitation, and do no more contribute to α.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

121

In contrast, solvent-exposed hydrophobic groups act as structure makers, resulting in a decrease in the water density around hydrophobic groups.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

122

In this case α is negative, and the temperature coefficient, dα/dT, is positive.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

123

This picture adopted here for proteins, also evolves from studies of volumetric properties of inorganic and organic electrolytes and small model compounds such as hydrophilic and hydrophobic amino acids (see ^{refs. 41,42}).

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

124

Our data for RNase A in H₂O and D₂O indicate high apparent thermal expansion coefficients of the protein and steeply decreasing slopes in their temperature dependence.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

125

Thus, they classify RNase A as a protein with a significant number of hydrophilic side groups at the protein surface.³⁰

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

126

Even much steeper slopes of dα/dT are found for proteins with more charged side groups, such as SNase³¹.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

Volumetric behavior and protein stability in D₂O

127

Water deuteration quite generally displaces the transition temperature of unfolding, T_m, to higher values (Table 1).^17–21

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

128

Intuitively, this temperature shift is attributed to a higher stability of the native protein in D₂O. However, the transition temperature only signals the equality of the Gibbs free energy of the native and unfolded state which results from enthalpy-entropy compensation.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

129

By a more detailed analysis of entropy and enthalpy behavior, some authors have concluded that at low temperatures the native protein is destabilized by D₂O.^20,21

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

130

Guzzi et al²¹. have argued that at the microscopic level this destabilization in D₂O originates from a less efficient solvation of solvent-exposed apolar side groups.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

131

For RNase A in D₂O, both the absolute value of the thermal expansion coefficient at low temperatures, as well as its temperature coefficient, are markedly higher than in H₂O (Table 1).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

132

In particular, the large isotope effect on dα/dT is notable.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

133

These volumetric effects of H₂O–D₂O substitution are in the same direction as obtained for proteins with a significant number of charged groups, for instance SNase,³¹ and also observed by addition of kosmotropic co-solvents such as glycerol or sorbitol.³⁰

Type: Result | Advantage: None | Novelty: None | ConceptID: Res14

134

This evidences a prime role of the enhanced solvation of the hydrophilic amino acid groups by D₂O, and clearly points towards a stabilization of the native form by D₂O. This would also be in agreement with the value of the Gibbs free energy of unfolding at room temperature, ΔG°(25 °C), deduced from the calorimetric data by assuming that the heat capacity change upon unfolding is independent of temperature.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

135

In that case, ΔG°(25 °C) values of 37 and 46 kJ mol⁻¹ K⁻¹ are obtained in H₂O and D₂O, respectively.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res15

136

Finally, the volume change, ΔV, that accompanies the unfolding in D₂O, is 40% lower than in H₂O (Table 1).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

137

A considerable part of this reduction may arise from a strong temperature dependence of ΔV, because as T_m increases, ΔV decreases significantly.^48,49

Type: Result | Advantage: None | Novelty: None | ConceptID: Res16

138

It might, at least partially, also be due to a more compact state of RNase A in the unfolded state, however.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res16

Protein stabilization as inferred from dielectric relaxation data

139

An intriguing question is, whether the increased hydration and stabilization in D₂O seen in the thermal and volumetric experiments leads to a change in the protein fold.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp2

140

Our strategy is to compare the isotope effect upon the protein relaxation time, τ₁, with the isotope effect upon the bulk viscosity, and also to compare the amplitudes S₁ of the protein peaks in H₂O and D₂O, respectively.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met3

141

Both types of information can be converted into structural data: τ₁ contains information on the hydrodynamic radius of the protein, and S₁ permits the calculation of the protein's dipole moment.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met3

142

We have conducted these experiments for RNase A and, for comparison, also for ubiquitin.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met3

143

As noted, the relaxation time τ₁ reflects protein tumbling.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met3

144

This process is considered to be a prototypical example for a process controlled by the hydrodynamic friction of the solvent,^32,33 and can be described by Debye's equation for the rotation of a dipolar sphere in a surrounding medium of viscosity η³⁸V_hydr is the hydrodynamic volume of the protein, a the hydrodynamic radius, and k_B is Boltzmann's constant.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

145

Because large deviations from spherical shape are needed to invalidate eqn. (6),³⁸ this relation is also applicable to RNase A, which can be pictured as an ellipsoid with an axial ratio of about 1.5.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

146

The viscosity of the buffer solutions were found to differ less than 1% from those of pure H₂O, η_H = 0.894 × 10⁻³ kg m⁻¹ s⁻¹ at 25 °C, and of pure D₂O, η_D = 1.101 × 10⁻³ kg m⁻¹ s⁻¹, respectively.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

147

Thus their ratio is η_D/η_H = 1.232,²⁴ where the subscripts H and D stand for H₂O and D₂O, respectively.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

148

Table 3 shows that, within experimental error, this ratio is indeed observed for the bulk water relaxation times τ₄.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

149

For the protein relaxation times,τ₁, this ratio is significantly smaller.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

150

Before discussing this isotope effect in detail, we compare the hydrodynamic radius deduced for RNase A with estimates from other sources.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

151

The most direct comparison is possible with ¹⁵N magnetic relaxation data.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

152

Magnetic relaxation monitors protein tumbling, as described by correlation functions associated with second rank (l = 2) spherical harmonics.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

153

Dielectric relaxation refers to first rank (l = 1) spherical harmonics.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

154

In the diffusive limit of single-particle reorientation, we have τ₁ = 3τ_NMR.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res17

155

From ¹⁵N relaxation data for RNase A by Cole and Loria,⁵⁰ we then estimate τ₁ = 20 ns and a ≅ 1.90 nm, in comparison with τ₁ = 27 ns and a ≅ 2.10 nm actually observed.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res17

156

The ifference is beyond experimental uncertainty, but one may note that the applied conversion, τ₁ = 3τ_NMR, holds only for processes reflecting single-particle motions.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res17

157

For many simple systems, including water, collective reorientational motions cause the dielectric relaxation time τ₁ to differ substantially from 3τ_NMR.⁵¹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

158

We have shown elsewhere³⁵ that protein-protein dipolar correlations lead to a concentration dependence of τ₁ which can account for the discrepancy of dielectric and NMR data.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

159

In all cases, the hydrodynamic radii are larger than the radii estimated from structural data.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

160

Molecular mass–volume correlations for globular proteins, for example given in ^{refs. 40 and 54}, predict an intrinsic radius of bare RNase A of 1.54 nm only (a thermal contribution of about 1 nm may be added).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

161

Similar discrepancies between hydrodynamic and structural radii are found for other proteins as well,^32,33 and have been commonly attributed to a shell of hundreds of water molecules contributing to the hydrodynamic radius of the protein.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

162

A common assumption is that these water molecules are bound on the time scale of protein reorientation, i.e. in the microseconds time regime.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

163

This result is highly inconsistent with results of NMR exchange studies^13,52,53 and MD simulations,^54,55 which clearly show that residence times of water molecules at protein surfaces are only on the sub-nanosecond time scale.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

164

The apparent differences between hydrodynamic and structural radii can be removed by computing the friction tensors from the protein shape on an atomic scale,^56,57 and by assuming a non-uniform solvent viscosity near the protein surface.⁵²

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

165

Turning to the effect of H₂O–D₂O substitution, we find the viscosity ratio of η_D/η_H = 1.232 to be reflected by the ratio of the relaxation times τ₄ of bulk water, but not by the ratio of the protein relaxation times τ₁ of RNase A and ubiquitin.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs12

166

There are two options for rationalizing this observation.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

167

First, as already noted, one expects a non-uniform solvent viscosity near the protein surface.⁵²

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

168

Thus, one possible explanation may presume that, owing to differences in hydration, the local isotope effect η_D/η_H differs from that in the bulk solvent.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

169

Alternatively, one may rationalize the observed behavior by assuming that, upon H₂O–D₂O substitution, the hydrodynamic radii of RNase A and ubiquitin shrink by about 5%.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

170

Two observations indicate that the isotope effects may indeed reflect a change in the hydrodynamic radius, and that this effect results from changes in the protein fold.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

171

First, it has been found in an elaborate small-angle neutron scattering (SANS) study⁴³ that the radius of gyration of lysozyme decreases from R_g = 1.38 nm in H₂O to 1.24 nm in D₂O. The order of magnitude of this effect (11%) is even larger than that observed here.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs13

172

In SANS, the contrast at the protein–water interface is higher than obtained by small-angle X-ray scattering (SAXS), and the radius of gyration essentially refers to the bare protein.⁴³

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

173

Thus, the change in the radius of gyration may well result from a change in the protein fold.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

174

In a more indirect way, such an effect has also been deduced from phosphorescence lifetime studies of several proteins.²⁵

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

175

Second, an interpretation in terms of a more compact fold in D₂O is consistent with the behavior of the amplitudes S₁ of the protein tumbling mode, which provide values for the effective dipole moments, μ_eff, of the proteins in solution (which differ from the dipole moments of the isolated proteins).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs14

176

The evaluation of S₁slightly depends on details of the dielectric model.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs14

177

For example, the Onsager–Oncley model^32,33 yields values of 276 D and 175 D (1 D = 3.30 × 10⁻³⁰ C·m) for RNase A and ubiquitin, respectively.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

178

Such high dipole moments are typical for proteins.^32,33

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

179

Any model yields, however, the same expression for the amplitude ratio where ρ is the number density of the protein molecules.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

180

Table 3 indicates that μ_eff,D is by about 5–10% smaller than μ_eff,H, indeed indicating a more compact state of the protein in D₂O, in which the charge distribution is spread over a smaller space than in H₂O.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

Conclusions

181

H₂O–D₂O substitution has been found to be an effective tool for singling out hydration effects on the thermodynamic behavior of proteins and their stability.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

182

In particular, the volumetric behavior reflected by the apparent thermal expansion coefficient, α, and its temperature coefficient, dα/dT, changes drastically upon isotopic substitution.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

183

These changes are intrinsically related to hydration properties and the interplay between hydrophilic and hydrophobic groups at the protein–water interface.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

184

The volumetric data clearly point toward a stabilization of the native form of RNase A in D₂O.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

185

An assessment of the underlying molecular processes can certainly not been done by considering the thermal and volumetric properties alone.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

186

As noted by Finney,⁴ in studying the subtle effects associated with protein hydration, as many techniques as possible should be applied.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

187

Here, we have complemented the DSC and PPC experiments by dielectric relaxation experiments for RNase A and ubiquitin.

Type: Method | Advantage: None | Novelty: None | ConceptID: Met1

188

These experiments yield evidence that the stabilization of the native form in D₂O reflects a more compact fold.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

189

A more compact fold in D₂O follows also from SANS data for another protein, lysozyme.⁴³

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

190

Presumably, the stronger solvation of hydrophilic groups and less effective solvation of hydrophobic groups at the protein surface cause the protein to reduce the solvent-exposure of the hydrophobic groups.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

191

These changes, in particular for enzymes with highly flexible surface groups, may very well alter the protein function in D₂O.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

Introduction

Methods

Samples

DSC and PPC experiments

Dielectric relaxation

Results and data evaluation

DSC results

PPC results

Dielectric relaxation data

Discussion

A scenario for the volumetric behaviour

Volumetric behavior and protein stability in D2O

Protein stabilization as inferred from dielectric relaxation data

Conclusions

Volumetric behavior and protein stability in D₂O