Solvent viscosity effects on protein dynamics studied by ultrasonic absorption

Solvent viscosity is known to play an important role in the kinetics of biochemical reactions, and has been suggested to modulate the dynamic structure of proteins. The effect of viscous cosolvents, of various molecular sizes, on the apparent ultrasonic absorption of bovine serum albumin in solution, at 37 degrees, has been measured in attempt to investigate the following phenomena: 1) The predicted modulating effect of viscous cosolvents on the "internal friction" of proteins, and 2) Possible differences between the microscopic and macroscopic pictures of the solvent viscosity concerning the proposed effect. We have found that A) The absorption of ultrasound (3-17 MHz) by the protein increases with increasing the cosolvent concentration. B) That increase correlates with the solvent viscosity for small cosolvent molecules, but not with macromolecular cosolvents, and C) Dextran solutions with the same concentration by weight, reveal similar ultrasonic absorption, in spite of large differences in their viscosity. A possible explanation is discussed.     

Analytical ultracentrifugation in a Gibbsian perspective

The analytical ultracentrifuge has come into new intensive use following complete instrumental redesign and the use of advanced computer technologies for the analysis and interpretation of experimental results. Major attention is now devoted to the evaluation of interactions between similar and dissimilar biological macromolecules in dilute and concentrated systems. Electrostatically charged biological solute systems additionally comprise low molecular weight charged and non-charged cosolvents. Solvent/cosolvent interactions, insufficiently considered in most current analytical ultracentrifugation analyses, may quantitatively affect solute/solute interactions. For comprehensive analysis the Svedberg derivation considering a buoyant molar mass (1 - rho0 partial specific volume)M2 and valid at vanishing solute concentration for strictly two component systems only, should be replaced, following classical thermodynamic analysis, by the ratio (delta rho/delta c2)(mu)/d pi/dc2 of the density increment at constant chemical potential of diffusible cosolvents, to the derivative of the osmotic pressure with solute concentration. Disregard of the solvent/cosolvent and solute/cosolvent interactions should be avoided.     

Effects of cryoprotectants on enzyme structure

The interaction between organic cosolvents and proteins is considered, especially from the point of view of effects on protein stability. It is concluded that each protein-cosolvent system constitutes a unique situation, making generalized predictions of expected effects difficult. Two classes of cosolvents are distinguished, based on the nature of their interactions with the protein surface. The thermodynamic instability to the system introduced by the presence of the cosolvent can be accommodated (i) by preferential exclusion of the cosolvent from the vicinity of the protein, (ii) by major structural changes of the protein, or (iii) by aggregation. Polyols tend to undergo preferential exclusion due to unfavorable interactions with nonpolar surface groups, whereas monohydric alcohols and other more hydrophobic cosolvents may undergo preferential exclusion due to adverse interactions with charged groups on the protein surface. Typical cosolvent effects on the structural and catalytic properties of enzymes are illustrated with data for ribonuclease and beta-lactamase with alcohol cosolvents. The relative hydrophobicity of the cosolvent is the major determinant of the effect of a cryosolvent on the enzyme stability and properties. Thus the position of the unfolding transition in cryosolvent will be decreased more by a more nonpolar cosolvent. Different cosolvents can have significantly different effects on the catalytic and structural properties of the same enzyme. Conversely the same cosolvent can have significantly different effects on similar proteins. The number and distribution of the nonpolar and charged groups on the protein's surface probably are the major determinants of the protein contribution to the solvent-protein interaction. The large temperature dependence of the rates of protein unfolding and refolding can be beneficially utilized in cryoprotectant studies of living cells.     

Effect of temperature on viscosity properties of some alpha-amino acids in aqueous urea solutions

Viscosities for solutions of glycine, DL-alpha-alanine, DL-alpha-amino-n-butyric acid, DL-valine, DL-leucine and L-serine in 5 mol kg(-1) aqueous urea have been determined at 278.15, 288.15, 298.15 and 308.15 K. The viscosity B-coefficients for the amino acids in the aqueous urea solution have been calculated at different temperatures. The effect of temperature on the B-coefficients is discussed on the basis of the Feakins equation. The contribution of solute to the activation parameters (delta mu0*2, deltaH0*2, deltaS0*2) for viscous flow of the solution have been calculated, together with the Gibbs energy, enthalpy and entropy of transfer for the amino acids from the ground-state solvent to the hypothetical viscous transition state solvent. The contributions of the charged end group (NH3+, COO-) and CH2 groups of the amino acids to B-coefficient and delta mu0*2 have been also estimated using the linear correlations between B-coefficient or delta mu0*2 and the number of carbon atoms in the alkyl chains of the amino acids. All the activation parameters are discussed in terms of the solute-solvent interactions in the ground and transition states.     

Viscosity dependence of O2 escape from respiratory proteins as a function of cosolvent molecular weight.

Laser photodissociation of respiratory proteins is followed by fast geminate recombination competing with escape of the oxygen molecule into the solvent. The escape rate from myoglobin or hemerythrin has been shown previously to exhibit a reciprocal power-law dependence on viscosity. We have reinvestigated oxygen escape from hemerythrin using a number of viscous cosolvents of varying molecular weight, from glycerol to dextrans up to 500 kDa. In isoviscous solutions, the strong viscosity dependence observed with small cosolvents is progressively reduced upon increasing the cosolvent's molecular weight and disappears at molecular weights greater than about 100 kDa. Thus, viscosity is not a suitable independent parameter to describe the data. The power of the viscosity dependence of the rate coefficient is shown here to be a function of the cosolvent's molecular weight, suggesting that local protein-solvent interactions rather than bulky viscosity are affecting protein dynamics.     

Solvent modulation in hydrophobic interaction chromatography

Solvents play a critical role in hydrophobic interaction chromatography (HIC), since the separation of proteins by HIC is based on the hydrophobicity of the proteins presented to the solvents. This review first describes the solvent properties which determine the effect of cosolvents on the binding and elution of proteins in HIC; i.e., the protein solvent interactions and the surface tension of water/cosolvent mixture. Second are presented the various cosolvents which have been tested as facilitating binding or elution of the proteins. Last, some examples of solvent manipulation which resolved complex mixtures of proteins by HIC are reviewed.     

Microscopic viscosity and rotational diffusion of proteins in a macromolecular environment

The Stokes-Einstein-Debye equation is currently used to obtain information on protein size or on local viscosity from the measurement of the rotational correlation time. However, the implicit assumptions of a continuous and homogeneous solvent do not hold either in vivo, because of the high density of macromolecules, or in vitro, where viscosity is adjusted by adding viscous cosolvents of various size. To quantify the consequence of nonhomogeneity, we have measured the rotational Brownian motion of three globular proteins with molecular mass from 66 to 4000 kD in presence of 1.5 to 2000 kD dextrans as viscous cosolvents. Our results indicate that the linear viscosity dependence of the Stokes-Einstein relation must be replaced by a power law to describe the rotational Brownian motion of proteins in a macromolecular environment. The exponent of the power law expresses the fact that the protein experiences only a fraction of the hydrodynamic interactions of macromolecular cosolvents. An explicit expression of the exponent in terms of protein size and cosolvent's mass is obtained, permitting definition of a microscopic viscosity. Experimental data suggest that a similar effective microviscosity should be introduced in Kramers' equation describing protein reaction rates.     

Diffusive motions control the folding and unfolding kinetics of the apomyoglobin pH 4 molten globule intermediate

The sperm whale apomyoglobin pH 4 folding intermediate exists in two forms, Ia and Ib, that mimic transient kinetic intermediates in the folding of the native protein at pH 6. To characterize the nature of the kinetic barrier that controls the formation of the earliest intermediate Ia, we have investigated the effects of small viscogenic cosolvents on its folding and unfolding kinetics. The kinetics are measurable by stopped-flow fluorescence and follow a cooperative two-state model in the absence and presence of cosolvents. Small cosolvents stabilize Ia, but, by applying the isostability test to separate the viscogenic effect of the cosolvent from its stabilizing effect, we found that, in both folding and unfolding conditions, the apparent rate constant decreases when solvent viscosity increases. The unitary inverse dependence of the apparent rate constant on solvent viscosity indicates a diffusion-controlled reaction. This result is consistent with the hypothesis that folding of the apomyoglobin pH 4 intermediate obeys a diffusion-collision model. Additionally, the temperature dependence of the reaction rate at constant viscosity indicates that the formation of Ia is also controlled by an energy barrier. Linear free energy relationships show that the transition state of the U <==> Ia reaction is compact and buries 45% of the surface area that is buried in native apomyoglobin. We conclude that the transition state of the U <==> Ia reaction resembles that for the formation of native proteins; namely, it is dry and its compactness is closer to that of the folded (Ia) form than of the unfolded form.     

The effect of viscosity on the accessibility of the single tryptophan in human serum albumin.

When reactions take place with one of the reactants tied to protein matrix, movements along the reaction coordinate towards the transition state can become coupled to structural fluctuations of the protein matrix. This investigation aims to test the assumptions underlying the arguments supporting such a coupling. A coupling is allowed only if the activation barrier is high and broad enough as shown to be the case for the proton catalyzed isotope exchange at Trp-63 of lysozyme. In the present investigation the activation barrier for the same reaction has been lowered radically in an effort to show that the coupling, as measured by the dependence of rate on solution viscosity, will diminish and ideally vanish, despite the unchanged effects of cosolvents on the chemical activities of all the reactants. The isotope exchange rate at the indole nitrogen of the single tryptophan residue of human serum albumin was measured with UV. This residue is rigidly held to the protein surface and the solvent access, although restricted, corresponds to a partially exposed residue. As a consequence, the isotope exchange rates and the bimolecular quenching rate of fluorescence by acrylamide, also measured, are high. The experiments were carried out at pH 5.2 where the molecule is in the N-form and the exchange is catalyzed by OH- ions. The activation energy of the hydroxyl catalyzed reaction is 22 kJ lower than for the proton catalyzed process. Under these conditions the exchange rate is viscosity independent both in the case of glycerol and in ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of proteins: temperature, pressure and the role of the solvent

We focus on the various aspects of the physics related to the stability of proteins. We review the pure thermodynamic aspects of the response of a protein to pressure and temperature variations and discuss the respective stability phase diagram. We relate the experimentally observed shape of this diagram to the low degree of correlation between the fluctuations of enthalpy and volume changes associated with the folding-denaturing transition and draw attention to the fact that one order parameter is not enough to characterize the transition. We discuss in detail microscopic aspects of the various contributions to the free energy gap of proteins and put emphasis on how a cosolvent may either enlarge or diminish this gap. We review briefly the various experimental approaches to measure changes in protein stability induced by cosolvents, denaturants, but also by pressure and temperature. Finally, we discuss in detail our own molecular dynamics simulations on cytochrome c and show what happens under high pressure, how glycerol influences structure and volume fluctuations, and how all this compares with experiments.     

Ultrasonic absorption in tobacco mosaic virus and its protein aggregates

The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein assemblies exhibit specific structural fluctuations detected in ultrasonic experiments. As in spherical viruses, the fluctuations exhibited by the protein aggregates having a quaternary structure similar to that of the virion were modified in the virus by interaction with the RNA strand. It is unlikely that the origin for the observed effect is due either to: (1) the difference in local mobility of the segment 89 to 113 of the polypeptide chain in TMV and in the helical aggregate on the one hand, and in smaller aggregates, on the other hand; or (2) a local fluctuation associated with proton transfer reactions or ion-pair interactions. The most remarkable feature in the TMV system is the fact that the two-ring disk showed no excess of ultrasonic absorption with respect to the A-protein oligomer, while a large increase of ultrasonic absorption was observed in the rod-like aggregate that had undergone the disk-helix transition.     

Viscosity scaling and protein dynamics

The rates of molecular motions in the interior of some proteins were found to scale with an inverse power of the external solvent viscosity. The data were explained by a flexible protein structure whose dynamics is partially controlled by the solvent. Reaction dynamics in the presence of structural fluctuations with finite lifetimes lead to a dynamic friction coefficient defined by a generalized Langevin equation and a fluctuation-dissipation theorem. A model for the dynamic friction is derived assuming that the fluctuation spectrum at the reaction site involves two components: solvent-independent diffusion of local structural defects in the protein matrix and global fluctuations coupled to the solvent. The theory is applied to the viscosity dependence of molecular oxygen-binding rates in sperm whale myoglobin.     

Biopharmaceutical formulation

The rapid maturation of the field of biopharmaceutical formulation is the result of the simultaneous development of a thermodynamic mechanism for protein-solvent interaction and identification of natural chemicals employed by nature to stabilize proteins in response to environmental stresses. In general, these cosolvents are excluded from the protein surface. Proteins are maintained in their native folded conformations by these cosolvents as a result of the highly unfavorable interaction between cosolvents and peptide backbones, which would be exposed to the cosolvent upon unfolding.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Effects of chaotropic and kosmotropic cosolvents on the pressure-induced unfolding and denaturation of proteins: an FT-IR study on staphylococcal nuclease

FT-IR spectroscopy was used to study the effects of various chaotropic and kosmotropic cosolvents (glycerol, sucrose, sorbitol, K(2)SO(4), CaCl(2), and urea) on the secondary structure and thermodynamic properties upon unfolding and denaturation of staphylococcal nuclease (Snase). The data show that the different cosolvents have a profound effect on the denaturation pressure and the Gibbs free energy (DeltaG(o)) and volume (DeltaV(o) change of unfolding. Moreover, by analysis of the amide I' infrared bands, conformational changes of the protein upon unfolding in the different cosolvents have been determined. An increase, a reduction, or an independence of the volume change of unfolding is observed, depending on the type of cosolvent, which can at least in part be attributed to the formation of a different unfolded state structure of the protein. The data are compared with the corresponding thermodynamic values of DeltaV(o) for the temperature-induced unfolding process of Snase as obtained by pressure perturbation calorimetry, and significant differences are observed and discussed.     

The effect of the solvent viscosity on the migration of small molecules through the structure of myoglobin.

The migration rate of small molecules through the structure of proteins can be monitored by quenching the light emitted from an excited optical probe located within the protein. In the present study we examined the influence of the solvent viscosity on the migration rate of the quencher anthraquinone sulfonate through myoglobin towards an excited Zn protoporphyrin molecule at the binding site of the protein. The solvent viscosity was increased by adding dextrans of different molecular weight but forming isoviscous solutions. The results demonstrate that the migration rate in the protein decreases with increasing solvent viscosity. This suggests that the fluctuations on the protein structure, which make the above migration possible, are affected by the solvent macroviscosity.     

Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of glycerol on hydrogen isotope exchange in lysozyme

Hydrogen isotope exchange rates for lysozyme in glycerol cosolvent mixtures [D. G. Knox and A. Rosenberg (1980) Biopolymers 19 , 1049–1068] have been analyzed as functions of solvent viscosity and glycerol activity in an attempt to determine which solvent properties influence protein internal dynamics. The effect of glycerol on the fast- and slow-exchanging protons is different. Slow-exchanging protons [H(t) < 20] are slowed by ever-increasing amounts as H(t) decreases. However, comparison with data for the effect of glycerol on the thermal unfolding of lysozyme [K. Gekko (1982) J. Biochem. 19 , 1197–1204] indicates that the large decrease in exchange rates for the slow protons is not consistent with a local unfolding mechanism of exchange. These effects are also too large to be easily rationalized in terms of solvent viscosity. Instead, we suggest that the large effect of glycerol on exchange of the slow protons is due to a “compression” of the protein, as a result of thermodynamically unfavorable interactions of glycerol with the protein surface. This reduces the protein void volume, which in turn decreases the probability of conformational transitions required for exchange of the slowest protons. Present data do not allow a distinction to be made between thermodynamic (glycerol activity) and dynamic (solvent viscosity) influences on exchange rates for the fast-exchanging protons, although the effect of glycerol on these protons is also probably too large to be consistent with a local unfolding mechanism. In this case, glycerol decreases the rate of catalyst diffusion within the protein matrix, either by decreasing the probabilities or amplitudes of “gating” reactions that allow passage of the catalyst from the solvent to the exchange site, or by increasing the relaxation times for these conformational rearrangements.