The role of protein-solvent interactions in protein unfolding

428

The role of protein-solvent interactions in protein unfolding Celia A Schiffer* and Volker D6tscht Protein unfolding occurs when the balance of forces between the protein's interaction with itself and the protein's interaction with its environment is disrupted. The disruption of this balance of forces may be as simple as a perturbance of the normal water structure around the protein. A decrease in the normal water-water interaction will result in an increase in the relative interaction of water with the protein. An increase in the number of interactions between water and the protein may initiate a protein's unfolding. This model for protein unfolding is supported by a range of recent experimental and computational data.

Addresses

* Department of Protein Engineering, Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA; e-mail: [email protected] t Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Correspondence: Celia A Schiffer Current Opinion in Biotechnology 1996, 7:428-432

© Current Biology Ltd ISSN 0958-1669 Abbreviations

GdnHCl guanidine hydrochloride MD molecular dynamics TFE trifluoroethanol

Introduction A protein remains folded as a result of a delicate balance of forces b e t w e e n the protein's interactions with itself and its interactions with its environment. W h e n the environment becomes denaturing (by heat, cold, pressure, acid, alkali, or organic denaturants) this balance is disrupted and the protein unfolds. Protein unfolding has been extensively reviewed and various models have been proposed [1-14]. We will not address the general issues of thermodynamics and equilibrium properties in protein unfolding or review the resulting models here. Instead, in this review, we will examine the role of water in the initial stages of the unfolding of a protein. "lb address the role of water in initiating protein unfolding we will examine recent computational and experimental studies. Molecular dynamics (MD) simulations suggest a simple picture of the role of water in the initial stages of protein unfolding. U n d e r a variety of denaturing conditions, water molecules c o m p e t e for the internal hydrogen bonds of a protein, disrupting its structure. T h e disrupted structure can then be further destabilized by specific interactions with a denaturant, or a denaturing bulk environment. Although indirect, since they are measured as averages over many molecules, experimental results support these conclusions.

Protein unfolding in atomic detail: computational studies What influences protein unfolding at the atomic level? T h i s question is difficult to answer experimentally, sincc all possible experinaents currently take average m e a s u r e m e n t s over many molecules. M D simulations that include explicit solvent can be used to look at the unfolding process in atomic detail, suggesting processes that cannot be directly obtained experimentally. T h e major shortcoming of this approach is that the timcscale of M D calculations are oF" the order of nanoseconds, whereas the timescale fi~r a protein to unfold, or a denaturant to diffuse in a solution, is of the order of milliseconds. Thus, m e t h o d s that accelerate the unfolding process must be used for an M D simulation to unfold a protein [9,14,15,16°,17]. Provided that direct comparisons to experimental data are made w h e n e v e r possible to avoid misinterpretation caused by acceleration artifacts, M D is still a powerful tool to probe protein unfolding. T h e role of water in unfolding a protein has been most extensively monitored in a series of computational studies on the unfolding of barnase [9,16°,18,19°]. T h e s e and other M D simulations that monitor the interactions b e t w e e n water and a protein (or p e p t i d e ) as the protein unfolds will be discussed. T h e most common way for calculations to acceleratc the protein unfolding process is to simulate t h e protein in water at temperatures well above the boiling point of water, which are thus no longer physically reasonable. [9,15,18,19",20-25]. With thc use of these high temperatures, it is unclear what the appropriate solvent density or pressure should be, and different laboratories have handlcd this in a variety of ways. T h e solvent density or the pressure of the system, however, will directly affect the e x t e n t to which hydrogen bonding b e t w e e n the water and the protein will occur. Thus, when the behavior of the water has been analyzed in detail, the results have not been consistent. Several protein simulations [17,22,26"] have observed a correlation b e t w e e n experimental hydrogen isotope protection factors derived by N M R and the percentage of simulation time for which water makes hydrogen bonds to particular sites on the protein. T h e s e correlations are reasonable, although not exact, since simulations usually follow the unfolding of a single molecule, whereas e x p e r i m e n t s average over many molecules. T h i s correlation, however, reinforces the notion that the interactions b e t w e e n water and the protein seen in M D calculations are reasonable. Many unfolding simulations have been performed on structured peptides. Since p e p t i d e s are smaller, their

The role of protein-solvent interactions in protein unfolding 8chiffer and D6tsch

unfolding and interactions with water can be analyzed in much more detail. T h e majority of such simulations have been performed on s-helical peptides [21,23,27,28,29",30], although recently a 17-residue 13-hairpin peptide from barnase was also studied [19"]. In most of the simulations of 0t-helical peptides, one or more water molecules were seen to insert between the turns of the helix. This type of water insertion was also seen and carefully analyzed [19"] in the 13 hairpin, where water competed for the peptide's hydrogen bonds and steadily unfolded the molecule as a function of temperature. Thus, water has been implicated in mediating the unfolding of both major forms of secondary structure. In the past year, several groups have used changes in pH to model protein (barnase) [31"] and peptide (amyloid [1-28] peptide) [29",30] unfolding in M D simulations. A change in pH is a physically reasonable alteration, as many proteins are known to unfold as a function of pH. Changing the p H is also computationally simple, as it only involves a change in the protonation state of the molecule. In addition, changes in the molecule can be observed under physically reasonable temperatures (barnase, 360K; amyloid ~ [1-28], 298K). In both sets of simulations, water was seen to play an active role in the conformational variability of the molecule. In the amyloid 13 peptide [29",30], a simulation at ' m e d i u m ' pH resulted in a distortion of the helix. This distortion forms a stable conformation in which the carboxylate side chain of a glutamic acid residue hydrogen bonds back to a serine and through two water molecules to the side chain of an aspartic acid simultaneously. Sequence substitutions [30] of these side chains in the simulations demonstrated that these results were indeed pH dependent. T h e acid denaturation of barnase [31"], for which water penetration was examined in greater detail, compares favorably with denaturation of the same molecule at high temperature [18]. In the later stages of unfolding barnase, clusters or chains of water molecules are seen to penetrate hydrophobic cores by hydrogen bonding to aromatic side chains. In both sets of calculations, water is seen to denature the secondary structure by replacing intramolecular hydrogen bonds. Two simulations [26",32] have also looked at protein unfolding with mixed solvents. Although adding co-solvents is often used experimentally either to denature or to stabilize a protein, representing co-solvents computationally is difficult as the diffusion rate of the solvents is slow relative to the simulation time. Nevertheless, the trends that these two studies highlight are consistent with what is seen experimentally. Alonso and Daggett [26"] compared the placing of a thermally denatured (498 K) structure of ubiquitin in 60% methanol at 335 K with the placing of the same structure in pure water at 335 K. In methanol, the molecule remained unfolded, whereas in pure water it began to refold. This study cannot properly address how methanol might cause a protein to denature, because

429

the molecule is already denatured when it is placed in the solution. However, in two parallel simulations on an s-helical peptide [28], one in pure water and the other in pure methanol, the methanol showed less hydrogen bonding to the peptide than did the water. It is thus unlikely that direct interaction with methanol breaks the hydrogen bonds of the peptide. In another study [32], a small 75-residue protein, equine infectious anemia virus Tat protein, was simulated in two parallel calculations at 300 K in pure water and in an aqueous mixture with 40% trifluoroethanol (TFE). From N M R studies, the structure of this molecule has been shown to form a stable three-helix bundle only in the presence of 40% T F E . T h e simulation shows that the molecule remains stable in 40% T F E , but in pure water the water molecules penetrate the c~ helices and begin unfolding the molecule.

Protein unfolding under more than one influence: experimental studies Experimentally, protein unfolding is not synchronized for all the molecules in solution. Thus, obtaining information about the exact molecular interactions between the protein and water or the protein and denaturants is very difficult. By simultaneously varying more than one environmental influence, recent experiments have shed light on what might occur at the molecular level. T h e s e experiments can be divided into two classes: those in which two types of denaturing conditions are applied simultaneously, and those in which a protein denaturant and a protein stabilizer are added simultaneously.

Compounding influences T h e denaturation of proteins by urea or guanidine hydrochloride (GdnHCI) has been studied as a function of pH [33,34]. Ribonuclease A, ribonuclease T1 [34] and barnase [33,35] are most stable to urea and G d n H C I denaturation near their isoelectric pH. At low pH, very low concentrations of the denaturants induce all three proteins to unfold. In addition, ribonuclease T1, which has its isoelectric point at about pH5.0, also unfolds more readily at high pH in the presence of low levels of denaturants. T h e enhanced susceptibility to denaturation of the proteins away from their isoelectric pH is likely to be due to an electrostatic repulsion of like-charged residues, that is, the protein's interactions with itself are less favorable. T h e denaturation of proteins by both urea or G d n H C I has also been studied as a function of temperature [36",37,38]. T h e temperature for unfolding ribonuclease T1 is 59.1 °C in the absence of GdnHCI, but is 40.6°C in 3.0M G d n H C I [38]. Similar trends were seen for barnase: as the concentration of urea was steadily increased, the unfolding temperature correspondingly decreased [37]. Agashe and Udgaonker [36"] monitored the thermal stability of the protein barstar at 15 different concentrations of G d n H C I and at 10 different temperatures in the range 273-323 K. T h e y found that increasing concentrations of GdnHC1

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Protein engineering

steadily destabilize the protein such that denaturation occurs more readily both with heat and cold.

Table 1

In all of the above cases, the combination of a denaturant (urea or GdnHCI) with another denaturing condition (temperature or pH) results in an additive effect that significantly decreases the stability of the protein molecule. Even though N M R studies have shown that urea directly interacts with proteins at low temperatures [39,40] this association is not observed at higher temperatures [40,41]. This implies that the denaturants are destabilizing the proteins in an indirect way.

Solute

Classification

Effect on protein

NaCO2CF3 Na2SO4 NaCI

Kosmotrope Kosmotrope Kosmotrope Kosmotrope Chaotrope Chaotrope Chaotrope Kosmotrope Chaotrope

Refolds Refolds Refolds Refolds Remains unfolded Remains unfolded Remains unfolded Refolds Remains unfolded

Competing influences

In concentrated G d n H C I and urea solutions [1,33], normal water structure no longer exists. T h e interaction between urea and water is weaker than that between water and water. This disrupts the dynamic network of hydrogen bonds that exists in pure water. Optical measurements using UV difference spectroscopy showed that the content of freely rotating water molecules is higher in mixtures of water with urea than in pure water [42]. Because of this weakening of the water-water interaction, the relative interaction of water molecules with other solutes is likely to be stronger. If a protein molecule is present, the water is more likely to compete for the internal hydrogen bonds of the protein, and thus initiate protein unfolding. If the above description of water molecules being disruptive agents that cause proteins to unfold is correct, then the addition of agents that order water should refold proteins. This is, in fact, what has been observed. In a set of N M R experiments performed on the unfolded DNA-binding domain of the 434 repressor in 7 M urea, a series of compounds were tested to see if the protein would refold [41,43°°1. T h e stabilization effect of the compounds used in the titrations closely follows the Hofmeister series [1,44,45]. In this series, cations, anions and neutral compounds have been ordered according to their effect on water structure, for example, by measuring an increase or decrease in the surface tension of the respective solutions. In the Hofmeister series nomenclature, molecules that strongly order water (such as NazSO 4 or glucose) are called kosmotropes and ones that interact weakly with water (urea or N a S C N ) are called chaotropes. T h e complete refolding of the DNA-binding domain of the 434 repressor in 7 M urea was achieved by the addition of a variety of kosmotropes, whereas when chaotropes were added, the protein remained unfolded (Table 1). This refolding of the protein in 7M urea cannot be explained by ions simply binding directly to the txrotein, since different anions have opposite effects on the protein, and osmolytes [46], such as glucose, also refold the protein. T h e s e results implicate the ordering of water as being crucial to maintaining a folded structure, as compounds that interact strongly with water seem to act by pulling water away from the protein thus allowing the protein to refold.

Effects of additives on unfolded 434 repressor in 7 M urea [41] and their classification in the Hofmeister series,

NHaCI NaSCN NaNa GdnHCI Glucose CH30H

Kosmotropes have also been seen to stabilize proteins against heat and pH denaturation. T h e addition of the kosmotrope KCI took both the completely unfolded, acid-denatured and the completely unfolded, alkalinedenatured [3-1actamase to a semifolded molten globule state [47]. Similarl.~; the addition of a series of osmolytes, which also act as kosmotropes, took the completely unfolded acid-denatured cytochrome c to a semifolded molten globule state [48]. In another case, the osmolyte sarcosine was shown to make ribonuclease A steadily more thermally stable at a range of pHs [49]. All these studies clearly show the proteins' structures becoming more ordered in the presence of kosmotropes. Pressure denaturation has also been shown to occur only when water is present. T h e stability of the Arc repressor under high pressure was measured as a function of increasing glycerol content [50]. As the percentage of glycerol in the solution was increased, the pressure required to denature the protein also increased• T h e authors extrapolated to the point where no water would be present and concluded that, in the absence of water, the protein was likely to remain folded, even under extreme pressure. T h u s the mechanism of unfolding by high pressure could also be that of increasing the number of water-protein interactions. What about protein?

the interaction

of urea with the

T h e role of urea in protein unfolding has been debated over the years in a variety of molecular models [4,51-54] on the basis of thermodynamic data. Urea has been seen by N M R to interact directly with the aliphatic side chains of a denatured protein [39,40]. Interactions with aromatic side chains have also been elucidated computationally [22,55,56]. This denaturant clearly interacts directly with the protein, but is this interaction the initiation event that causes protein unfolding? T h e effect of direct binding is likely to be small in initiating protein unfolding when compared with the chaotropic action of this molecule in disrupting the bulk water. Other chaotropes, such as NaSCN, can also denature proteins, but when used as the sole denaturant have been observed to cause the protein to precipitate more readily. Chaotropes such as urea and

The role of protein-solvent interactions in protein unfolding Sch#er and D6tsch

GdnHCI are then likely to serve a secondary function: by specifically binding to hydrophobic side chains, they help to keep an unfolded protein soluble once it is unfolded. This model of protein unfolding, in which water molecules actively participate in the disruption of protein structure, does not directly take thermodynamics into account. However, incorporating these considerations into a thermodynamic analysis could help to explain the discrepancies between the various thermodynamic unfolding models [4].

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Making use of the water penetration model In a recent computational study, the forces that cause a protein to unfold were directly probed [57"]. This was achieved by selectively changing the interaction potential between the water's interaction with the protein and the water's interaction with itself. A series of simulations at 300K were performed on the crystal structure of the DNA-binding domain of the 434 repressor and the resulting structure was monitored. The only condition that resulted in the molecule unfolding was an increase of 10% in the Coulombic interaction potential between the water and the protein. This 'active' water resulted in the protein beginning to unfold in a manner that was consistent with what is seen experimentally [39,41,43"•,58]. An 'active' water model mimics the tendency of water to disrupt protein structure under denaturing conditions and thus provides a useful tool as a physically reasonable method of unfolding proteins.

16. Karplus M, Sail A: Theoretical studies of protein folding and • unfolding. Curr Opin Struct Biol 1995, 5:58-73. A review of recent advances in computational protein unfolding and folding. 1Z

HLinenbergerPH, Mark AE, Van Gunsteren WF: Computational approaches to study protein unfolding: hen egg white lysozyme as a case study. Proteins 1995, 21:196-213.

18.

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Conclusions In this review; we have presented a range of experimental and computational data suggesting that the initiation of protein unfolding may be as simple as the perturbance of the normal protein-water interaction. This perturbance can be caused by temperature, pH, pressure or chaotropes. The result of this perturbance appears to be an increase in the affinity of water molecules for the protein. The water molecules then compete for the protein's intramolecular hydrogen bonds and initiate protein unfolding.

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MarkAE, Van Gunsteren WF: Simulation of the thermal denaturation of hen egg white lysozyme: trapping the molten globule state. Biochemistry 1992, 31:7745-7749.

21.

Tirado-Rives J, Jorgensen WL: Molecular dynamics simulations of the unfolding of an (z-helical analogue of ribonuclease A S-peptide in water. Biochemistry 1991, 30:3864-3871.

22.

Tirado-Rives J, Jorgensen WL: Molecular dynamics simulations of the unfolding of apomyoglobin in water. Biochemistry 1993, 32:4175-4184.

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Acknowledgements The authors thank WF van Gunsteren and K W~ithrich fur their support, and R Ward, R Kelly; AA Kossiakoff, AM de Vos, K Pearce, T Elkins and J Sohl for helpful suggestions.

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