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CHAPTER1 Noncovalent Forces Important to the Conformational Stability of Protein Structures Kenneth I? Murphy 1. Introduction Proteins are the workhorses of life, responsible for catalyzing the numerous chemical reactions necessary to living cells as well as being important structural molecules. The functionality of proteins requires the correct spatial placement of amino acid side chains and prosthetic groups that, in turn, requires a defined three-dimensional structure of the protein chain. The three-dimensional structure is obtained through the folding of the polypeptide chain from an ensemble of fairly loose, disordered con- figurations, collectively referred to as the unfolded state, into a well- defined compact configuration, known as the native state. There are three related questions that can be asked regarding a protein’s native structure: 1. What is it? 2. HOWi s it attained?a nd 3. Why is the native structuret he one observed? The first question is primarily addressedt hrough physical techniques, such as X-ray crystallography or multidimensional NMR spectroscopy. The second question is primarily addressed by kinetic studies aimed at assessing the pathway of folding. The third question, with which this chapter is concerned, is primarily addressed through structural thermodynamics, the study of the connection between a protein’s structure and the forces that maintain it. From: Methods in Molecular Slology, Vol. 40: Protern Stabrky and Folding: Theory and Practice Edited by: 6. A. Shirley Copyright Q 1995 Humana Press Inc., Totowa, NJ 1 2 Murphy Most of us have been taught in undergraduate biology and biochemis- try that proteins are stable because of the “hydrophobic effect,” a force that is entirely entropic and results from the restructuring of water around apolar surfaces. Recently it has become increasingly clear that this simple view of protein stability is inadequate to account for the experimental data. The emerging view is not yet completely clear and there seems to be considerable disagreement within the literature. Nevertheless, the points of agreement between various investigators, although not empha- sized in the literature, are many and will be discussed in this chapter. This chapter will address the following questions: 1. What are the primary forces responsible for the stabilization of globular proteins? 2. How can quantitative estimates of these forces be obtained?a nd 3. Can quantitative estimates of the various forcesb e relatedt o protein structures in a predictive manner? In the latter part of the chapter,w e will discuss an empirical approacht hat we have recently applied to estimating the thermodynamics of folding/ unfolding transitions and binding reactions from high resolution structures. This approach illustrates that even with the current, incomplete understanding of the noncovalent interactions in proteins, accurate predictions of the thermodynamics of processesi nvolving proteins are possible and the future for a structural energetic understanding of proteins is promising. 2. Energetic Description of Protein Folding/Unfolding Transitions The stability of a protein molecule customarily is described by the difference in free energy between the unfolded and native states, AG. Using the standard convention, a positive value of AG indicates that the native state is energetically favored over the unfolded state. The free energy difference, AG, is defined in terms of the enthalpy, AH, and entropy, AS, differences as: AG=AH-TAS (1) For the case of protein denaturation, AH and AS are dependent on temperature through the heat capacity difference, AC,, as: AH=AHR+ACp(T-TR) (2) and AS = AS, + AC, In (T I TR) (3) Noncovalent Forces in Protein Stability 3 where A& and A& are the enthalpy and entropy changes at the reference temperature, TR, and T is the absolute temperature in K. In the native state many of the protein functional groups are buried in the core, from which solvent water is excluded. Additionally, the amino acid side chains in the core are essentially fixed by specific interactions and tight packing. The peptide backbone is also relatively fixed through- out the protein. The amino acid side chains on the surface of the protein are also likely to have their motion somewhat restricted because of their close proximity to each other, although this restriction should be much less than that of the interior side chains. On unfolding, the interactions between protein groups within the core are disrupted and replaced with interactions with the solvent. Also, the backbone and side chains become much more mobile and, as discussed later, this gain in configurational entropy strongly favors the unfolded state. 3. Primary Forces Involved in Protein Stability Since this chapter will discuss our current, largely empirical, understanding of the role of various forces in stabilizing globular protein structures, a detailed physical description of those interactions will not be given here. A brief overview is, nevertheless, appropriate. There are many ways in which one can envision a partitioning of the forces involved in protein stability. Here we consider van der Waals interactions, hydrogen bonds and electrostatic interactions, configurational entropy, and the unique role played by water. 3.1. Van der Waals Interactions Van der Waals interactions, also known as London dispersion forces, result from transient dipoles that nonbonded atoms induce in each other. Such interactions, although ubiquitous, are fairly weak and short range. Because of the strong distance dependence of van der Waals interactions, the packing of atoms in the protein core, relative to their interaction with solvent, is important in determining whether they will stabilize or destabilize the native state. 3.2. Hydrogen Bonds Hydrogen bonds are noncovalent interactions that arise from the partial sharing of a hydrogen atom between a hydrogen bond donor group, such as a hydroxyl -OH or an amino -NH, and a hydrogen bond acceptor atom, such as oxygen or nitrogen. Many potential hydrogen bond donor Murphy and acceptor groups are present in proteins, namely the peptide backbone groups and the polar amino acid side chains. A hydrogen bond is essentially a dipole-dipole interaction, although it is often modeled as a charge-charge interaction between partial charges. Because hydrogen bonds involve permanent dipoles, the interaction also has an angular dependence.A thorough review of hydrogen bonding in proteins has been given by Baker and Hubbard (I), and the distribution of hydrogen bond patterns and their implications for folding also have been recently discussed by Stickle et al. (2). 3.3. Electrostatic Interactions Electrostatic interactions occur between charges on protein groups. Such charges are present at the amino- and carboxy- termini and on many ionizable side chains, Charges buried in the protein interior will interact strongly since the protein interior is considered a low-dielectric medium, but it has been suggested that such interactions may only play a minor role in protein stability since they are not overly common (3). Electro- static repulsion may be more important, not only in destabilizing the native state but also in terms of its effect on the degree of extension of the unfolded state (4-6). 3.4. ConfZgurationaZ Entropy Whereas the interactions discussed above tend to stabilize the native protein structure, configurational entropy destabilizes it. The gain in configurational entropy relates to the increased degrees of freedom avail- able to the protein chain in the unfolded state relative to the native state. This gain comes from both the side chains and the backbone. Although the peptide backbone of most residues in a globular protein is relatively fixed (i.e., has low entropy), those residues that are most buried within the core of the protein have even fewer backbone degrees of freedom. The entropic effect of burying side chains is more pronounced since they have considerable flexibility on the protein surface. As larger proteins bury more of their side chains, they will have an overall larger configurational entropy change per residue. This effect may help to set a limit on the size of a globular folding domain. The amino acid composition also affects the configurational entropy. For example, proteins containing a large proportion of proline residues will have a lower entropy in the unfolded state and thus will be more Noncovalent Forces in Protein Stability 5 stable. The opposite will be true for proteins containing a large proportion of glycine. 3.5. The Role of Water Water plays a crucial role in the stabilization of proteins. The small molecular size of water relative to other liquids, along with its complex hydrogen-bonded structure, make it an excellent solvent for many functional groups. These same features also give rise to the hydrophobic effect, The complex role of water in protein stability has been long recognized, but defining that role in quantitative terms is a topic of intense discussion more than 30 yr after Kauzmann’s seminal review (3). One of the difficulties in discussing the role of water in stabilizing proteins is simply the definition of terms. Many phrases, such as “hydration,” “ solvation,” and “the hydrophobic effect,” are in common use but are not clearly defined (see, for example, refs. 7-9). In particular, the term “hydrophobic effect” has been used to refer to: 1. The transfer of a compound from an organic liquid into water (9); 2. The transfero f apolars urfacef rom any initial phasei nto water (10,11);a nd 3. Any transfer into water accompaniedb y a large AC,, (7). In this chapter, “hydration” refers to the transfer of any group from the gas phase into water and “hydrophobic effect” refers to the transfer of apolar groups from any initial phase into water. It is important to bear in mind that this usage is purely operational. The hydrophobic effect does not refer only to the properties of water or imply a “purely entropic” effect. In this sense, the hydrophobic effect will be different for gases, liquids, and solids. Considering protein stability, the hydrophobic effect refers to the energetic consequences of removing apolar groups from the protein interior and exposing them to water. 4. Experimental Evaluation of Forces In attempting to assesse xperimentally the magnitude of the various contributions to protein stability, there are two basic approaches that can be taken: to study the stability of proteins as a function of various environmental variables, such as temperature, denaturant concentration, site- specific mutations, and so on; and to study model systems. In the study of model systems one attempts to mimic the unfolding process, but with a system simpler than a protein, which, hopefully, will be easier to inter- 6 Murphy pret. Since a comprehensive review of the literature of these areas is beyond the scope of the present work, we will present here only those results necessary to understanding the various, current viewpoints on the partitioning of energetic contributions. 4.1. Protein Studies In studying proteins the goal is to measure stability as a function of an environmental perturbant. Perhaps the most fundamental studies of protein stability involve temperature as the environmental variable. Differ- ential scanning calorimetry (DSC), in which the excess heat capacity of a protein solution is determined as a function of temperature, can provide all the thermodynamic parameters that specify the stability of the protein as a function of temperature: AH, AS, and AC, (for reviews see Chapter 9; refs. 12,13). 4.1.1. Differential Scanning Calorimetry Studies Numerous DSC studies have revealed several regular features regarding the thermal denaturation of globular proteins. Denaturation is always accompanied by a large positive ACp. The specific heat capacity change normalized to the number of residues in the protein, AC,, is directly proportional to the number of apolar contacts in the protein interior (13). (Overscored values below are always normalized to the number of residues). Likewise, AC, is proportional to the amount of apolar surface area buried in the protein. Although it is now clear that the exposure of polar surface makes a significant, negative, contribution to AC, (10,14-16), globular proteins generally bury a constant amount of polar surface area per residue (II, 17), and Ai$ can be considered to some degree as a measure of the “hydrophobicity” of the protein. For a large number of globular proteins, the specific enthalpy changes, u, when plotted as a function of temperature and taking Ac, to be independent of temperature, converge to a common value, G*, at a specific temperature, T*H (13). This behavior is revealed by plotting A?? at some convenient temperature, usually 298 K, vs A?$ for a set of proteins (18) in what has been called an MPG enthalpy plot (19). The slope of this plot is (298 - T*H) and the intercept is AH-* . Analysis of the set of globular proteins shown in Fig. 1 indicates a value of 373 + 6 K for T> and a value of 1.35 Z!I0 .12 kcal/(mol-res) for m* (IO). These values are independent of whether or not AC, is constant with temperature; they are Noncovalent Forces in Protein Stability 7 600 ACp / cal K“ (mol-res)-’ Fig. 1. Plot of m vs ACp for globularp roteins( adaptedfr om ref. 10). The slope of the line (298.15- TH*) gives a TH * value of 373 K. The intercept gives 1vi* equal to 1.35 kcal/mol. It should be noted that AH decreasesw ith increasing AC,, suggestingt hat the hydrophobic groups make a negative contributiontp the AH at 298 K. Data are from ref. 13, Table 1, with the correction that AC, for parvalbumin is 13.4 cal/W(mol-res) and AH for pepsinogeni s positive. simply reference values useful for describing the enthalpic behavior of proteins from -0-80°C. As discussed below, the convergence of the enthalpy terms is of fundamental importance in permitting an empirical calculation of energetic terms. Inspection of Fig. 1 leads to the somewhat surprising conclusion that @ decreases with increasing hydrophobicity. This is a key observation in interpreting the partitioning of the forces that contribute to protein stability, as will be seen later. Just as with the specific enthalpy changes, it is observed that the specific entropy changes also converge to a defined value, As*, at a specific temperature, T %. This convergence is readily seen inan MPG entropy plot (Fig. 2) in which AS at 298 K is plotted against AC,, (18). Here, the slope is In (298/Ts*) and the intercept, As*. T%h as the same value for all processes involving the transfer of apolar groups into water (18,20), 8 Murphy r....I....I....I...,r....l....l....I.... -‘*IO 11 12 13 14 15 16 17 18 ACp / cd Km’ (mol-res)-’ Fig. 2. Plot of As vs AC* for globular proteins (adapted from ref. 10). The slope of the line (In [298/Z’,*]) gives a T,* value of 385 K. The intercept gives Ag* equal to 4.3 cal W(mol-t-es). The negative slope is indicative of the negative AS associated with the exposure of apolar surface on unfolding. Data are from ref. 13, Table 1. 385 f 1 K. For globular proteins, the value of Ag* is 4.3 & O.l/cal W (mol-res) (13,18). As with AH, it is seen that AS decreases with increasing hydrophobicity of the protein. This is to be expected, since the exposure of apolar groups to water has long been known to be accompanied by a negative AS (21). Furthermore, Baldwin (20) observed that the transfer of apolar liquids into water is accompanied by a AS of zero at T:; hence, Ag* can be considered as the configurational entropy change accompanying the unfolding of globular proteins. The AS attributable to solvent restructuring, i.e., primarily the hydro_phobice ffect, is-thus given as ACP In (T/T”,). Interestingly, a plot of AG at 298 K vs ACP (not shown) shows essentially no correlation. In other words, the slope is zero and the intercept is just the average AG. This indicates that AG is practically independent of the hydrophobicity of the protein at 298 K. Noncovalent Forces in Protein Stability 9 The convergence behavior discussed above presents an intriguing puzzle in understanding the forces stabilizing proteins and has been addressedb y several investigators (10,13,18-20,22-25). Why do the specific enthalpy and entropy converge at some high temperature, and why do the specific values at 298 K decrease with increasing “hydrophobicity?’ Various interpretations of these phenomena will be discussed later. 4.1.2. Mutational Studies The ability to make single amino acid changes has provided another means by which investigators can probe the stabilization of proteins (26). The basic rationale is that the contribution of a specific interaction can be determined by adding or removing a side chain involved in that interaction and comparing the mutant stability to that of the wild-type protein. For example, a hydrogen-bonded serine can be mutated to an alanine, thus removing a hydrogen bond. The difference between the wild-type stability and the mutant stability, MG, is typically taken as the hydrogen bond contribution to the free energy. Similarly, values of U, AAS, and AAC, can be obtained. Shirley et al. have studied a number of hydrogen bond mutants of RNase Tl(27). On average, they find a AAG of 1.5 kcal/ mol for a hydrogen bond mutant. However, one must be cautious in the interpretation of such results, as can be seen by considering the effect of a mutation on the unfolding process. The overall hydrogen bond contribution to the energetics of unfolding can be represented in the following way. First, the hydrogen bond between the donor, D, and the acceptor, A, is broken in the interior of the protein: A*D+A+D (4) Then both A and D are transferred to solvent: ‘4 -+A, W ~-a, (5b) where the subscript aqi ndicates that the group is solvated in an aqueous environment. In the hydrogen bond mutant described above, the donor has been deleted and the remaining contribution of the hydrogen bond groups is represented by Eq. (5a). The difference between the wild-type 10 Murphy and mutant reactions corresponds to (A D + A + D,,) rather than to l (A D + A, + Daq)t he desired overall reaction. Furthermore, this simple l analysis assumes that the mutation has no other effects, such as structural rearrangements or bond strains. Shirley et al. (27) have pointed out that if a water molecule replaces the deleted donor, then the measured difference will be closer to the desired contribution. Even so, it cannot be assumed that the reaction A H,O + A,, makes no contribution to the energetics. The energetic l effects of site-specific mutations may be complex, even in the simple case where no structural rearrangements occur in the protein. Numerous mutation studies have also been directed at determining the contribution of the hydrophobic effect by, for instance, replacing large hydrophobic side chains with Ala or Gly (28-32). Interpreting the results of such studies is again difficult, which can be seen by replacing the hydrogen bond in Eq. (4) with a van der Waals contact. Matthews and coworkers (32) have recognized this complication and have included a “cavity” term to account for it. However, the flexibility of proteins can allow for repacking in response to a deletion, further complicating the problem. Lee (33) recently analyzed this problem in detail, showing that the maximum destabilization will occur for a rigid structure in which no repacking occurs. Most studies of the effects of mutations investigate only the changes in AG, and not in Lw, AS, and ACP. Although AG directly reflects the overall stability of the protein, because of the usually compensating effects of m and AS (34,35) it provides scant information on the actual contributors to the stability and may, in fact, be misleading. As an example, Connelly et al. (36) studied?a series of T4 lysozyme mutations in which seven different residues were substituted for Thr 157. The results showed that even though the changes in AG are relatively small and regular, the changes in m (and hence AS) are large and reveal no trends. If a trend in AG arises from some specific interaction, for example the hydrophobic effect, then all other thermodynamic functions should be corre- lated with that same interaction. It is also important to note that mutations may affect both the native and unfolded states of a protein. This possibility was addressed early in mutational studies (37,38) and is the subject of a recent review by Dill and Shortle (39).