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.     

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.     

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.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

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.     

Second virial coefficient studies of cosolvent-induced protein self-interaction

Protein self-interaction is important in protein crystal growth, solubilization, and aggregation, both in vitro and in vivo, as with protein misfolding diseases, such as Alzheimer's. Although second virial coefficient studies can supply invaluable quantitative information, their emergence as a systematic approach to evaluating protein self-interaction has been slowed by the limitations of traditional measurement methods, such as static light scattering. Comparatively, self-interaction chromatography is an inexpensive, high-throughput method of evaluating the osmotic second virial coefficient (B) of proteins in solution. In this work, we used self-interaction chromatography to measure B of lysozyme in the presence of various cosolvents, including sucrose, trehalose, mannitol, glycine, arginine, and combinations of arginine and glutamic acid and arginine and sucrose in an effort to develop a better fundamental understanding of protein self-interaction in complex cosolvent systems. All of these cosolvents, alone or in combination, increased B, indicating a reduction in intermolecular attraction. However, the magnitude of cosolvent-induced changes in B was found to be largely dependent on the ability to control long-range electrostatic repulsion. To the best of our knowledge, this work represents the most comprehensive virial coefficient study to date focusing on complex cosolvent-induced effects on the self-interaction of lysozyme.     

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.     

Studies of effects of macromolecular crowding and confinement on protein folding and protein stability

In cells, proteins execute specific tasks in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement or excluded volume plays an important role in its stability and dynamics. In this article we present results of our experimental and theoretical investigations of the confinement and macromolecular crowding effects on protein. On the experimental side we study the stability of encapsulated cytochrome c against unfolding induced by the presence of denaturants, such as urea. Results show that, as the pore size in which protein is trapped is reduced, protein shows higher stability against denaturant-induced unfolding. On the theoretical side, after reviewing our previous study of the confinement effects on the equilibrium and dynamic properties of protein using a minimalist (two-dimensional lattice, Monte Carlo, Brownian dynamics) model, we have extended the model so that the effects of macromolecular crowding on such properties can be studied. Our simulations show that both folding and unfolding times increase with the number of crowders in solution, however, the equilibrium constant is affected such that the equilibrium is shifted towards the folded state. Furthermore, our results show that, for a fixed number of crowders as the size of crowder (or excluded volume) increases, the average size of protein at equilibrium decreases.     

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.     

Enthalpies of DNA melting in the presence of osmolytes

The melting of DNA in the presence of osmolytes has been studied with the intention of obtaining information about how base pair stability is affected by changes in solution conditions. In previous investigations, the melting enthalpies were assumed to be constant as osmolalities change, but no systematic evaluation of whether this condition is true has been offered. This paper presents calorimetric data on the melting of two synthetic DNA samples in the presence of a number of common osmolytes. Poly(dAdT)*poly(dTdA) and poly(dGdC)*poly(dCdG) melting have been examined by differential scanning calorimetry in solutions containing ethylene glycol, glycerol, sucrose, urea, betaine, PEG 200 and PEG 1450 at increasing osmolalities. The results show small, but significant changes in the enthalpy of melting of the two polynucleotides that are different, depending on the structure of the cosolvent. The polyols, ethylene glycol, glycerol, PEG 200 and also urea all show decreases in melting enthalpy, while betaine and sucrose display increases with increasing concentration of cosolvent. The large stabilizing PEG 1450 shows no change within the experimental errors. Using concepts relating to preferential interactions of the cosolvents with the DNA base pairs, it is possible to interpret some of the observed changes in the thermodynamic properties of melting. The results indicate that there is strong entropy-enthalpy compensation upon melting base pairs, but entropy increases dominate to cause the decreases in stability with increased cosolvent concentration. Excess hydration parameters are evaluated and their magnitudes discussed in terms of changes in cosolvent interactions with the DNA base pairs.     

Probing protein-sugar interactions

We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.     

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin.

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.     

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.     

Effects of thermodynamic nonideality on protein interactions. Equivalence of interpretations based on excluded volume and preferential solvation

Published results on the stabilization of proteins by sucrose (J.C. Lee and S.N. Timasheff, J. Biol. Chem. 256 (1981) 7193) have been reexamined and interpreted in terms of thermodynamic nonideality. The composition dependence of activity coefficients may be accounted for on a statistical-mechanical basis using the concept of excluded volume. An expression is derived in which the effect of sucrose on determination of the partial specific volume of a protein, previously interpreted in terms of preferential protein solvation, is also seen to be attributable to excluded volume. Gel chromatographic studies of the reversible unfolding of alpha-chymotrypsin are presented which demonstrate temperature- and sucrose-mediated changes in the effective volume of the enzyme. These measurements support the quantitative interpretation of the stabilization in terms of thermodynamic nonideality arising from the difference between covolumes for sucrose and the two isomeric states of alpha-chymotrypsin. By establishing the equivalence of the two approaches that have been used to account for the effects of inert solutes on protein transitions, the present investigation eliminates the need for any distinction between such solutes on the basis of molecular size; and also enhances greatly the potential sensitivity of thermodynamic nonideality as a means of probing protein isomerizations, since greater displacement of the equilibrium position may be effected by small rather than by macromolecular solutes present at the same weight concentrations.     

Refolding SDS-denatured proteins by the addition of amphipathic cosolvents

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the α-helical or the β-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.     

Using high-performance liquid chromatography to measure the effects of protein-stabilizing cosolvents on a model protein and fluorescent probes

The effect of cosolvents on the fluorescence of solutes was measured manually and in an automated high-performance liquid chromatography (HPLC) system that eliminates fluorescent contaminants on-line. The HPLC system was used to show that the effect of cosolvents on the fluorescence spectrum of heated chymotrypsin (a measure of unfolding) correlates with the effect of the solutes on the heat stabilization of catalytic activity; r2=0.73 with 12 example cosolvents. Changes in the fluorescence of model probes showed that known counteracting solutes slightly decrease the polarity of the solvent. Different cosolvents affect the proton transfer indicator, 2-naphthol (a model for tyrosinyl residues) differently, polyhydric alcohols enhance the protonated naphthol emission whereas zwitterionic solutes enhance naphthoxide fluorescence. The results with the automated system are consistent with the known stabilizing effects of the cosolvents and validate it as a tool to explore the development of novel cosolvents and their effects on multiple biological systems.     

Interaction of selected cosolvents with bovine alpha-lactalbumin

Alpha-lactalbumin constitutes about 3% of bovine milk proteins. The preferential solvent interactions between selected cosolvents (sorbitol, sucrose and glycerol) and alpha-lactalbumin at pH 7.5 was determined using precision densitimetry. The preferential interaction parameter (xi(3)) and other thermodynamic parameters were calculated at different solvent concentrations. The xi(3) parameter was maximum at 30%, 45% and 40% (w/v) concentrations with the values of -0.282g/g, -0.171g/g and -0.299g/g for sorbitol, sucrose and glycerol, respectively. Thus the principal driving energy in the system being preferential hydration and mutual exclusion of bulk solvent. There was only a marginal change in the CD spectra of the protein with these cosolvents indicating the integrity of secondary structures. The results of thermal denaturation measurements indicated an increase in thermal stability of alpha-lactalbumin with these cosolvents. In the presence of 30% sorbitol there was an increase in the apparent thermal transition temperature (apparent T(m)) from 65 to 71 degrees C. These results indicate that the selected cosolvents in this study stabilizes alpha-lactalbumin without altering the structure of the protein.     

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.     

Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides

Protein stability and solubility depend strongly on the presence of osmolytes, because of the protein preference to be solvated by either water or osmolyte. It has traditionally been assumed that only this relative preference can be measured, and that the individual solvation contributions of water and osmolyte are inaccessible. However, it is possible to determine hydration and osmolyte solvation (osmolation) separately using Kirkwood-Buff theory, and this fact has recently been utilized by several researchers. Here, we provide a thermodynamic assessment of how each surface group on proteins contributes to the overall hydration and osmolation. Our analysis is based on transfer free energy measurements with model-compounds that were previously demonstrated to allow for a very successful prediction of osmolyte-dependent protein stability. When combined with Kirkwood-Buff theory, the Transfer Model provides a space-resolved solvation pattern of the peptide unit, amino acids, and the folding/unfolding equilibrium of proteins in the presence of osmolytes. We find that the major solvation effects on protein side-chains originate from the osmolytes, and that the hydration mostly depends on the size of the side-chain. The peptide backbone unit displays a much more variable hydration in the different osmolyte solutions. Interestingly, the presence of sucrose leads to simultaneous accumulation of both the sugar and water in the vicinity of peptide groups, resulting from a saccharide accumulation that is less than the accumulation of water, a net preferential exclusion. Only the denaturing osmolyte, urea, obeys the classical solvent exchange mechanism in which the preferential interaction with the peptide unit excludes water.