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DENl\TURED STATES OF PROTEINS

Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

Ken A. Dill Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

David Shortie Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 KEY WORDS:

unfolded states, conformational change, molten globules, acid denaturation, solvent denaturation.

CONTENTS PERSPECTIVES AND OVERVIEW. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

795

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

796

THEORETICAL FRAMEWORK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G ood Solvents, P oor Solvents, and Theta Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . Heteropolymer Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EXPERIMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solvent Denatured States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH and Thermally Denatured States . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . Experimental Models o f Denatured States Under Physiological Conditions . . . . . . . . . . S UMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

800 803 803 807 807 813 817 820

PERSPECTIVES AND OVERVIEW Because the biological properties of proteins arise primarily from their native confonnations, considerably more attention during the past

60 years has been

focused on the native confonnations than on the non-native confonnations. In

the past kw years, however, there has been increasing interest in non-native and denatured states of proteins since these less ordered states play important roles in at least three important phenomena: 1.

Protein folding and stability.

The denatured states of proteins are equal in importance to the native states in determining the stability of a protein, since stability is defined as the dif­ ference in free energies between native and denatured states. In cells and 795

0066-41 54/9 110701 -0795$02.00

Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

796

DILL

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SHORTLE

tissues the native states are in dynamic equilibrium with the denatured con­ formations (1-3). 2. Transport across membranes. To cross a lipid bilayer after translation and folding, some proteins must first be converted into a partially folded or denatured conformation , either spontaneously or with the help of "unfoldases" (4-6). 3. Proteolysis and protein turnover. Numerous studies have implicated the denatured state as the primary target for degrada­ tive enzymes, both in states of stress such as heat shock, and under physiolog­ ical conditions (7-9). In this review, we summarize results from theory and experiment that bear on the following questions: What are the denatured conformations of pro­ teins? How are they affected by external conditions, such as solvent composi­ tion, temperature, pH , and ionic strength? In what way do these denatured conformations depend on the amino acid composition and specific sequence of the protcin? A brief review of the forces that determine conformations in polymers leads to the concepts of "good," "poor ," and "theta" solvents. The denatured state is not a single entity microscopically or macroscopically. Rather , theory and experiments indicate that the denatured state should be viewed as a distribution of many microstates that change with the conditions of solution and with the protein sequence. Although classical experiments by Tanford (1) showed that several proteins in 6M guanidine hydrochloride (GuHCl) exhibit the hydrodynamic properties of random coils, this result has often been misconstrued to imply that the denatured state is a random coil under all conditions . There is now evidence that, even in 6M GuHCI, proteins can have significant amounts of internal structure . Under the much broader range of conditions that are typically used to denature proteins, recent theory and experimental data suggest that de­ natured proteins are often very compact, with persistent hydrophobic cluster­ ing and considerable residual secondary structure . The traditional view holds that the native state is determined by the amino acid sequence and that the denatured states are not, but current evidence indicates that the primary sequence is also a major determinant of denatured conformations . Perhaps the most important denatured state, defined here as Do, is that which is in equilibrium with the native molecule under physiological conditions. Although no experiment has directly observed this state, model systems consisting of peptides and shortened proteins may lead to a greater un­ derstanding of the properties of Do. INTRODUCTION Whereas the native state of a protein has a relatively well defined set of atomic coordinates , the denatured states do not, and must be described within a different framework, involving conformational ensembles . We therefore be-

Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

DENATURED STATES OF PROTEINS

797

lieve it is important to begin a discussion of denatured states with definitions of some terminology. First, we distinguish between "denatured" and "un­ folded" states as follows. The term "denatured" was first given an operational definition (1) to designate " . . . a major change from the original native structure . . . " that is noncovalent, cooperative, and reversible, in principle if not in practice . [ "Denaturation" is sometimes used to refer to a variety of poorly understood irreversible alterations in protein structure that include aggregation , disulfide bond rearrangement, and backbone cleavage (10), but we do not use that meaning here.] We use "unfolded" state to refer to a specific subset of denatured states, namely conformations that are highly open and solvent-exposed, with little or no residual structure; such states are generally obtained only under strongly denaturating conditions . A second subset of denatured states is referred to here as "compact denatured" states , obtained under weaker denaturing conditions. Whereas "denatured" and "un­ folded" have often been used interchangeably, we believe experimental reso­ lution has now progressed to the stage that it is useful to divide the category of denatured states into two parts: unfolded and compact. In this review, the term "state" has its usual thermodynamic meaning; it refers to an ensemble of the many different configurations of individual molecules , the average of which corresponds to a given macroscopically observable quantity. We denote this macroscopic parameter, or "reaction coordinate," as a from the native state to the denatured state. For example, a might be the molecular radius as measured by intrinsic viscosity or by scattering experiments . We usually use the term "state" more narrowly as an abbreviation to mean "stable equilibrium state ," i . e . an ensemble of con­ figurations at a minimum of free energy. For example , consider the radius r as the reaction coordinate. The observed radius in an equilibrium population (see Figure 1) represents a large number of different configurations ( i . e . an "ensemble") of the many molecules in solution, because: (a) a chain can configure in many different ways to have radius r = r*, corresponding to the equilibrium state, and (b) other chain configurations with r =1= r*, will also be populated in accord with the Boltzmann distribution law. A "two-state sys­ tem" has two minima along the reaction coordinate. It follows that some sort of free energy barrier must separate these two minima. This review is not concerned with the nature of this barrier, or with the kinetics of folding; for a recent review of these topics , see Kim & Baldwin (12). If the conformational free energy, dG (a, x), of a protein chain also depends on an external parameter, x, such as temperature or concentration of some denaturing agent, then external changes of x can lead to a transition, as shown in Figure 1. By "cooperatiive ," we mean that the state of the protein (as represented by the average < a» is observed to have a sigmoidal dependence on x (for example, see vs x in Figures lA-D). By sigmoidal , we refer only to the S-shaped

798

DILL & SHORTLE

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Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

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a' �j£x '12 defines a poor solvent. For nonpolar amino acids in water, it is estimated from oil/water partition ex­ periments that X 25°C (19, 47). Hence water is an extremely 3.37 at T poor solvent for polypeptide chains which, like most naturally occurring proteins, contain from 25 to 50% nonpolar amino acids. For a heteropolymer, it is not X alone that predicts whether the solvent will expand or contract the polymer. Instead the quantity X4>2, where 4> is the fraction of residues in the chain that are nonpolar, is the approximate predictor of chain expansion or contraction. The net result is that, even though 6M GuHCI reduces X by only about 25% (20, 47), this is sufficient to cause an aqueous solution of 6M GuHCI to be a good solvent for most proteins. Significantly low­ er concentrations of GuHCI (i.e. 0-4 M) will be poor solvents for most proteins (47). Therefore, for a given set of conditions, proteins with many nonpolar residues will have denatured states that are more compact than will proteins with few nonpolar residues. The heteropolymer theory pre­ dicts that a protein at its isoelectric pH should denature if X exceeds a particular value, XO, irrespective of whether X is caused to increase by temperature or by denaturants. Thus at the midpoint temperature, the con­ formations of a thermally denatured molecule should closely resemble those of a molecule denatured by GuHCI or urea (at the midpoint denaturant con­ centration). Proteins are typically denatured by GuHCI in the range of I to 4 M; for most proteins these are poor 30lvent conditions. Therefore, in the absence of charge effects, denaturation either by heating to high temperatures, on the one hand, or by GuHCl or urea, on the other hand, should lead to a relatively compact denatured state (at the transition midpoint) compared to a chain in a ==

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DENATURED STATES OF PROTEINS

805

theta solvent. Moreover, further increase in denaturant (i.e. either of tempera­ ture or of denaturing agent) beyond the denaturation midpoint should lead to further gradual increase of the radius as the solvent becomes "better," as shown in Figures 3 and 4. Figures 3 and 4 also show that the radius of the denatured states at the denaturation midpoint depends on the fraction of nonpolar residues in the chain. The theory predicts that weakly denatured proteins, either by temperature or by low denaturant concentrations, have considerable numbers of contacts among hydrophobic residues. Another property is predicted by theory for polymer chains in poor sol­ vents: compact chains should have some internal structure: helices, sheets , and turns (21,22). The amount of structure should increase with compactness (21,22) and will also depend on specific interactions . Structure arises because 20r-�--�--�--, 0.38 15 0.38 V' Vn



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Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

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DENATURED STATES OF PROTEINS

807

the severe steric constraints in relatively compact chains are selective for certain configurations, including helices, sheets , and turns. "Local" in­ teractions among neighboring residues in the chain also contribute to structure in denatured states. EXPERIMENTS

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Solvent Denatured States As emphasized above, the properties of denatured states depend on the solvent conditions. The denatured state under physiological conditions, Do, is the most relevant to an understanding of protein stability, since stability is the free energy difference, .::lG Gnative - Gdenatured RT In [N)/[Dol . Unfortunately, since most proteins under native conditions are stable by + 5 to +20 kcal/mole, then only one out of every 104 to 1015 protein chains is in the Do state. Consequently, experimental studies have concentrated on D states under conditions quite different than aqueous buffers at neutral pH and 20° to 37°C. By analyzing how the properties of denatured states vary under these more extreme conditions , estimates of the properties of Do can often be made by extrapolation back to physiological conditions . Experimental studies of denatured states have also been hampered by two major practical obstacles. The most serious of these is that the denaturing conditions used in an experiment are often those that also lead to limited solubility, so it is often necessary (but very difficult) to distinguish between denatured and precipitated protein. In addition, until recently, the physical methods for characterizing the structure of denatured proteins have only provided information at low levels of resolution. Intrinsic viscosity , gel filtration, and quasi-elastic light scattering can determine the molecular radius (23-25) , and various types of spectroscopy can monitor the amount of secondary structure and the environments around tyrosine and tryptophan residues (23, 26, 27) . Important as they are for characterizing low-resolution structure, these methods provide relatively little insight into the molecular details of those conformations. Fortunately, high-resolution NMR spectros­ copy has enormous potential for providing this type of structural information (for a recent review, see reference 28). A problem of long-standing interest has been whether the denatured states that arise fmm heating as opposed to the addition of denaturants are different (29, 30 , 1). Heat denaturation may be something of a misnomer, since heating a protein to high temperatures at its isoelectric pH has not been widely used for reversible denaturation experiments because it often leads to irreversible covalent changes ( 1, 10) . Thus thermal denaturation generally refers to heating to moderate temperatures at low (or high) pH. In these cases, the role of electrostatics is probably very important. Therefore, these processes are =

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808

DILL

& SHORTLE

considered in the next section on pH denaturation rather than in the present section. The most widely used strong denaturants are GuHCI and urea, which lower protein stability in proportion to their concentrations (3,31,32). Although the chemistry of interaction between proteins and aqueous solutions of urea and guanidine is not fully understood,most available evidence indicates that these compounds cause water to become a better solvent for the nonpolar amino acids (33, 34); i.e. they weaken the hydrophobic interaction. As mentioned above, Tanford observed that 6M GuHCI is a good solvent for proteins and it is "universal" in the sense that the radius, as determined by intrinsic viscosity and light scattering measurements, depends only on chain length, r = (constant)nO.67, and not on the amino acid sequence, for more than 10 different proteins (1, 2); see Figure 5. Early evidence supported the conclusion that, in high concentrations of urea or guanidine hydrochloride, denatured proteins are highly unfolded and have little residual structure (35-39, 1). On this basis, denatured states of proteins have often been assumed to resemble random coil conformations. However, computer simulations show that the radius of random-flight chains is not a particularly sensitive measure of partial clustering of the chain (40). Therefore the Tanford experiment does not rule out the possibility that denatured proteins may havc partial structure in 6M GuHCI. Recent studies on a number of proteins using spectroscopic methods have turned up un­ ambiguous evidence of residual structure, in some cases even at very high denaturant concentrations. For example, Bierzynski & Baldwin (41) used NMR spectroscopy to characterize GuHCI-denatured ribonuclease A at low pH and found evidence that the a-helix that corresponds to the C-peptide is present a significant fraction of the time. Haas et al (42,43) used fluorescence energy transfer between two extrinsic probes to measure the distance between amino acids 1 and (49 + 53) in reduced ribonuclease A in 6M GuHCl. They observed an average distance significantly less than that predicted for a random coil polypeptide. A similar fluorescence transfer study by Amir & Haas (44) of reduced bovine pancreatic trypsin inhibitor (BPTI) in oM GuHCI suggests that approximately 50% of molecules exhibit native distances be­ tween residues 1 and 26, while in the other 50% of the molecules these two residues are separated by a much greater distance. Since this persistent structure was greatly diminished in SAM GuSCN, Amir & Haas proposed that the highly hydrophobic chain segment between the donor-acceptor pair forms structure that persists in 6M GuHCI but is nearly eliminated under still stronger denaturing conditions. More specific evidence from high-resolution NMR a:so shows the per­ sistence of structure in strong denaturants. Wishart (45) labeled the alpha

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the radius, r, is proportional to nO.67 in 6M GuHCl; thus this is a good solvent.

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Annu. Rev. Biochem. 1991.60:795-825. Downloaded from www.annualreviews.org by University of Tennessee - Knoxville - Hodges Library on 02/17/13. For personal use only.

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DILL & SHORTLE

carbons of the nine glycines of Escherichia coli thioredoxin with eJ3. While all nine peaks could be resolved in the native state, five or six peaks were still resolved in 9M urea at room temperature. Since in 2 .5M GuHCI at 65°C all the resonances collapsed into three broad bands, the authors concluded that significant local structure must persist in 9M urea to give rise to the observed chemical shift differences . Similarly, the proton NMR spectrum of BPTI in 8M urea shows clear evidence for some residual structure (46). Another probe of denatured conformations is the amount of solvent expo­ sure of the amino acid residues, which can be estimated from the dependence of protein stability on denaturant concentration. Thermodynamic and statisti­ cal mechanical models (34, 47) indicate that m, the rate of change of the free energy of denaturation with respect to the denaturant concentration, is approx­ imately proportional to the difference in solvent accessible surface area A between the D and N states . If the proportionality constant for this relation­ ship were known, Ad (exposed nonpolar area of the denatured state) could be determined from m, since An (exposed area of the native state) can be calculated from the X-ray crystal structure. The heteropolymer theory (47) and experimental evidence (2, 32) suggest that Ad for a typical protein is considerably less than that predicted for a fully exposed chain. A large number of experiments on a variety of proteins (reviewed in reference 3) show that m is almost invariably a constant, displaying little if any dependence on denaturant concentration over the small concentration ranges that are typically accessible experimentally; see Figure 6. An approximate linearity over nar­ row ranges of denaturant concentration is expected from theory (34, 47); but whether there are small deviations from linearity has not yet been fully resolved. Recent data strongly support the idea that replacing single amino acid residues can affect the denatured states of a protein . For different mutant forms, m can either increase or decrease relative to the wild-type value (32, 48-55) . Small changes in m of either sign can be accounted for in part by significant changes in the area exposed in the denatured state, Ad (47) . However, some single-site replacements are observed to lead to very large changes in m (48) that cannot be accounted for by the heteropolymer theory (47). For staphylococcal nuclease, a multiple mutant has been identified that increases mGuHCI by almost a factor of two (D. ShortIe, A. M. Meeker, unpublished observations), suggesting that the wild-type D state must retain a large number of residues in a solvent-excluded configuration. For several proteins, increasing the charge on the protein by increasing or decreasing pH can also lead to very significant increases in murea (56, 57). Presumably, the fraction of buried surface exposed upon denaturation is increased by electrostatic repulsion when the polypeptide chain is highly charged.

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DILL & SHORTLE

To analyze the contribution of the large hydrophobic residues of staphylo­ coccal nuclease to the stability of the native state and to the residual structure that persists in the denatured state, ShortIe et al (58) mutated individually each of the leucine, valine, isoleucine, phenylalanine, tyrosine, and methionine residues to both alanine and glucine. After overexpressing and purifying 83 single mutant proteins, GuHCl denaturation was used to determine mGuHe[ and ilGH,a. the stability in the absence of denaturant. Perhaps their most important finding was the existence of a statistically significant correlation between the absolute value of the change in mGuHe[ away from that of wild-type and a loss in stability. From this result, they made the argument that part of the stability loss for some mutants must be a consequence of the altered o state structure reflected in the change in mGuHe[. On analyzing the changes in stability in this collection of mutant nucleases, ShortIe et al (58) found it very difficult to account for various patterns in their data solely in terms of changes in native state interactions. For example, if the principal effects of shortening aliphatic side chains were to decrease the free energy of hydrophobic group transfer on folding and/or to generate cavities in the native structure, one might expect that the stability loss for a series of substitutions at one position would show a linear relationship with the number

of carbon atoms (or methylene equivalents) removed. But at more than half of the 29 positions analyzed, a twofold or greater variation was found among the changes in stability that accompanied rcmoval of methylenes from leucine, isoleucine, valine, and mathionine. At some positions, the gamma, delta, and epsilon- carbons made a greater contribution to stability than did the beta carbon, whereas at other positions the reverse was found. In addition, at several positions the stability loss that accompanied removal of the beta carbon exceeded the 2.0 to 2.5 kcallmole upper limit estimated for the contribution of methyl groups to the cohesion of hydrocarbon solids. ShortIe et al proposed that some of these unexpected effects could be a consequence of disruption of the hydrophobic clustering in the denatured state predicted on thc basis of changes in mGuHCl. For mutant nucleases with a less structured denatured state, part of the stability loss would then be accounted for by the larger value of the entropy gain on denaturation. Flanagan et al (59) have recently used small-angle scattering of both X-rays and neutrons to monitor the denaturation of staphylococcal nuclease in urea. Both the radius of gyration and the density of local clustering of the polypeptide chain in the denatured state have been determined. Perhaps their most surprising finding was the bilobed shape of the denatured state around 3M urea (conditions where only 10% of molecules remain in the native state) . This structure gradually expanded and became more nearly spherical with increasing denaturant concentrations. Data collected on several mutant nucle­ ases confirmed earlier suggestions that single amino acid substitutions can significantly alter the residual structure in the denatured state (48, 23).

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DENATURED STATES OF PROTEINS

813

A few rdatively unstable proteins, such as alpha-lactalbumin (76), carbon­ ic anhydrase (62), bovine growth hormone (62), and beta-lactamase from Staphylococcus aureus (63), which are denatured in low concentrations of GuHCl, show transitions more complex than those between two simple fixed states. Although such behavior has been attributed to three states-the native state, the unfolded state, and a compact denatured state (60)-it may also be consistent with a two-state mechanism in which the denatured state changes continuously from a compact form to an unfolded form with increasing denaturant concentration (see appendix and Ref. 13). Additional evidence is needed to unequivocally establish the existence of a discrete third species , or equivalcntly to establish that there is a free energy barrier between the compact and unfolded denatured states for these transitions in GuHCI. Whereas urea and GuHCl are among the most widely used agents for denaturation of proteins, nearly all organic solvents that are miscible with water also act as protein denaturants. At low concentrations (5-20%), simple aliphatic allcohols promote the breakdown of protein structure, presumably by direct association of the hydrophobic component of the solvent with exposed hydrophobic residues of the denatured protein (64, 65). The efficacy of alcohols allld similar compounds in promoting denaturation is markedly de­ pendent on both their concentration and the temperature. Elevated tempera­ tures increase denaturation, whereas low concentrations of some compounds, such as methanol and ethanol, can stabilize the native state around O°C (64). At higher concentrations of organic solvent, which lead to aggregation and precipitation of many proteins, the appearance of a more structured state has been noted (66). Interpretation of the optical rotary dispersion (ORD) and circular dichroism (CD) spectra of this state suggests the polypeptide chain is driven into a conformation with large amounts of alpha-helical structure (66). For example, trifluoroethanol is commonly used to promote alpha helix formation in small peptides (67). pH and Thermally Denatured States

Since proteins invariably contain basic and acidic residues, they possess a net charge which increases as the solution becomes more acidic or more basic relative to the isoe1ectric pH. In the absence of ion-pairing, the electrostatic free energy of a charged protein is rcduced upon denaturation because charge repulsion is reduced as charges are distributed over the larger volume of the denatured molecule. Unlike the case with solvent denatured states, the persistence of large amounts of residual structure in acid-denatured states has been simple to demonstrate, especially in the presence of salt (68-71). The most direct evidence was that of Aune et al (36), who performed two parallel ex­ periments. In the first, they denatured three different proteins (ribonuclease ,

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& SHORTLE

lysozyme, and chymotrypsinogen) at pHs from 1.65 to 2.18 using tempera­ tures between 56°C and 60°C and then tested the nature of the resultant denatured state by further treatment with increasing GuHCI concentrations . They observed a small cooperative transition, which they interpreted as evidence that the low-pH thermal denaturation process had not fully unfolded the protein. In a parallel experiment, they denatured the protein in 6M GuHCI (at pH 4 . 4 and 6 . 7), followed by heating. In this experiment, they observed no cooperative transition and therefore concluded that denaturation in 6M GuHCI leads to a less structured denatured state than thermal denaturation at low pH. More recent experiments have confirmed that thermal unfolding under acidic conditions often leads to a compact denatured state, sometimes referred to as the A state (72-74) . At pH below 4.0, dramatic changes are observed in the environment of buried aromatic residues in alpha-lactalbumin as moni­ tored by CD spectroscopy and UV absorption, whereas indicators of second­ ary structure such as CD in the far ultraviolet and infrared spectroscopy detect only minor structural changes (75, 76) (see Figure 7A). This acid-denatured form, termed the A state, remains quite compact, with gel chromatography, small-angle X-ray studies, and quasi-elastic light scattering all suggesting that the polypeptide chain has increased its radius from approximately 18 A in the N state to 20 A in the A state (29, 77). Perhaps the most convincing evidence that the A state is not a relatively subtle alteration in the N state comes from studies of its thermal stability. No cooperative transition, only a monotonic decrease, was observed in the CD Signal as the temperature was raised for molecules in the A state (76, 78). In addition, no appreciable heat absorption was detected by scanning calorimetry (79). Recent NMR studies by Baum et al (80) revealed that the proton spectrum of the A state is very different compared to either the native or the urea­ denatured states. For some of the resolvable protons (shown in Figure 7B), tentative assignments were made by using magnetization transfer from the native state under conditions where N and A are in rapid exchange. The four aromatic residues in the N state with the largest chemical shifts away from the random coil values also had large chemical shifts in the A state, suggesting that some of the residual structure may be native-like. This conclusion was supported by hydrogen-exchange measurements that revealed that some of the slowly exchanging protons in the A state are also among the most slowly exchanging in the N state. In particular, amide protons of residues 89 to 96 exchanged 50 times more slowly than expected for a random coil. Since this chain segment forms an alpha helix in the N state, it may also be helical in the A state. Because protection from hydrogen exchange was not found for other amide protons that form alpha helices in the N state, Baum et al proposed that a hydrophobic kernel is the most prominent structure in the A state (80) .

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B

A

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Wavelength (nm) Figure 7

I I , , , I t, I I, I '\I , , , I t, I I. I I I , I , , , I I , I , , , I I I I , , , I I I I I , I , , I I I I , , , v-

240 260 280 300 320

Wavelength (nm)

A-STATE



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>-3



U-STATE

en

o 'T1

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9.0

8.0

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A. Far (a) and near (b) UV circular dichroism spectra of three states of alpha· lactalbumin: N(- - - -), A (- - -), and D ( ....), D is the urea-den atured form (109). B. Low field regions of the proton NMR spectra for the three states of alpha-lactalbumin (80).

where A is the acid-denatured fonn and

@ Z

en

00 VI

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816

DILL & SHORTLE

Carbonic anhydrase (81 , 62), beta-Iactamase (82), cytochrome c (83, 84), apomyoglobin (84), staphylococcal nuclease (85), and several other proteins have all been shown to g ive rise to compact states on aci d denaturation. Kuwajima has listed six properties characteristic of the A state of a­ lactalbumin (60): (a) secondary structure similar to the native structure, (b) solvent inaccessibility of some residues (tryptophans) that are also buried in the native structure, (c) small expansion of the radius relative to the native state less than or equal to 10% (i.e. a 30% increase in volume), (d) propensity to aggregate, perhaps due to exposure of hydrophobic residues, (e) no thermal transition upon heating (although there is some cooperativity upon GuHCI denaturation), (j) slow but extensive intramolecular fluctuations. Kuwajima refers to this as the "molten globule" state. The term "molten globule" originated with Ohgushi & Wada (83) . Although it originally referred only to the acid-denatured A state, the term molten globule has now come to refer to compact states that may also arise from other conditions of denaturation, for example by thermal or GuHCI denaturation (60, 74, 77). "Globule" refers to the native-like compactness, and "molten" refers to the higher enthalpy and entropy relative to the native structure, as in the melting of a solid, and to the larger structural fluctuations , particularly in the side chains (77), relative to the native structure. Because there are some differences in definition of molten globule among groups, we favor the recommendation of Kim & Baldwin (12) that the term "molten globule" be used to designate the theoretical model of non-native states of proteins that have "native-like" topology described by Ptitsyn (74) and Shakh­ novich & Finkelstein (86, 87); that "compact denatured states" refer to experimentally observed compact non-native statcs; and that the A state refer specifically to acid-denatured compact states. Fink et al (73, 82, 88) have shown, particularly by CD spectra taken in the near and far UV, that the acid-denatured state may not be u nique, and that for different proteins , these states may have little, moderate, or large amounts of residual structure that may or may not change continuously with salt concentration. Recently, Fink and colleagues (88) have provided important new informa­ tion on acid-denatured states for several different proteins. Characterization of the efficacy of different salts in stabilizing the A state relative to the unfolded state clearly suggests that charge neutralization, not indirect solvent effects, is the dominant role of added anions (88) . In addition, proteins that denature directly to the A state at low pH without added salt and proteins that do not denature even at pHs below 1. 0 develop a salt requirement for stability of the A state when urea is added. The data of Fink et al can be explained in terms of a balance between electrostatic repuls ion among residues of like charge and the forces that act to constrain and structure the polypeptide chain. Some proteins (i.e. alpha-lactalbumin) tend to require little or no salt to collapse into

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DENATURED STATES OF PROTEINS

817

the A state, whereas others (i.e. beta-Iactamase) often require high con­ centrations of salt to first reduce electrostatic repulsion. An extreme example of the latter effect can be seen in histone H I , which spontaneously denatures at very low salt concentrations even at neutral pH (89). The existence of two stable populations of denatured states at low pH, separated by a free energy barrier, has been predicted by addition of elec­ trostatics contributions to the heteropolymer theory (90) . Low pH denatura­ tion can lead to both an open unfolded state, and a compact denatured state. As shown in Figure 8, the predictions are in good agreement with the pH-ionic sltrength phase diagram of myoglobin of Goto & Fink (73). Accord­ ing to the theory, the appearance of two stable denatured states is a conse­ quence of a balance of four forces. The open denatured state is stabilized by the electrostatic force, which leads to expansion, balanced by chain elasticity, which opposes expansion. Similarly, the compact denatured state is stabilized by the hydrophobic interaction, which leads to contraction , balanced by the excluded volume entropy, which opposes contraction. States intermediate in radius are slightly less stable, because either charge repulsion drives them to expand or hydrophobic interactions drive them to contract. The heteropolymer model predicts two very different mechanisms of de­ naturation. On the one hand, denaturation of charged molecules can lead to a transition from N to either of two stable denatured states in equilibrium: one unfolded, and one compact with a small free-energy barrier between them. On the other hand, for denaturation near the isoelectric pH either by heating or by denaturants, the heteropolymer model predicts only a single sharp transition from native to a compact denatured state, which then gradually unfolds with further increase in temperature or denaturant. In support of this theory, experiments of Pfeil (29) show some similarity between temperature- and solvent-denatured alpha-lactalbumin compared to the acid-induced denatured state. Experimental Models of Denatured States Under Physiological Conditions

To develop better experimental models for Do, the denatured state in equilib­ rium with the native structure under physiological conditions, it is necessary to characterize molecules at the margin of stability. Only unstable molecules have significant populations of denatured conformations. One approach is to study the p:roperties of short peptides, which can often be analyzed in aqueous solutions at neutral pH and ambient temperature, and therefore promise to be more representative of the Do state. Early efforts to detect persistent secondary structure in short peptides with sequences corresponding to alpha helices or beta turns of known proteins (91-93) were unsuccessful in all cases but one (94). More recent work,

DILL & SHORTLE

818

0.3 A

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0.2

-



N

0.1

---

..c:



0>

c

a

Q)

t.... .......

CI)

u c

2

UA

4

3

5

35 A 0.3

high density u nfolded

0

0.2

folded

0.1 o

low density u nfolded

2

3

Figure 8 Phase diagram of apomyoglobin vs pH and ionic strength. (a) Experiments of Goto & Fink (73) showing three states: native (N), acid unfolded (VA)' and compact denatured (A). (b) Predictions of the heteropolymer theory with electrostatics (90). Iso-radius lines on the left identify the possible phase boundary between VA and A.

however, has provided a number of examples in which synthetic peptides, free in solution, can adopt the same secondary structure as a segment of a known protein with the same amino acid sequence. The most extensively studied example is the C-peptide, which corresponds to an alpha helix formed by the first 1 3 residues of ribonuclease A. Generated by cyanogen bromide

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DENATURED STATES OF PROTEINS

819

cleavage, this protein fragment exhibits approximately 30% helical structure at O°C by CD (94). Baldwin and colleagues have studied extensively synthetic peptides with altered amino acid sequences in order to determine the origins of the unusually high stability of this short helix (95, 96). They conclude that, in addition to the favorable helix propensities of the residues, salt bridge formation and an aromatic interaction between side chains, plus favorable interactions of terminal charged groups with the helix dipole, all contribute to helix stability (97). Similarly , synthetic peptides corresponding to an alpha helix in BPTI (98), the C helix of myohemerythrin (99), residues 132-153 of sperm whale myoglobin (100) , residucs 50-61 of ribonuclease A (101), and a proteolytic fragment of bovine growth hormone ( 102) all form alpha helices in solution. None of these helices, however, are as stable as that formed by the C- peptide. A short synthetic peptide corresponding to a highly immunogenic segment of the influenza hemagglutinin molecule has been demonstrated to form a rela­ tively stable type II beta tum in solution, although this sequence in the native protein does not form a tum (103). In several short peptides containing three or more hydrophobic residues, the protons on the hydrophobic side chains have been found by NMR spectroscopy to exhibit significant chemical shift dispersion, a result consistent with aggregation or clustering of these neigh­ boring hydrophobic groups ( l 04-106). It appears that isolated peptides in solution under native conditions can have a propensity to form native-like structures. This propensity to form structure can be further enhanced if protein sub­ structures are appropriately constrained by disulfide bonds or hydrophobic contacts. One example is a peptide model of parts of BPTI studied by Oas & Kim (107). A disulfide-bonded pair of peptides corresponding to 30 of the 56 residues of BPTI forms a structure in solution that is similar to that of native BPTI (107). One peptide corresponds to a two-turn helix and a short beta strand, whereas the other has the sequence of parts of two antiparallel beta strands. Th:! CD spectra and the aromatic region of the proton NMR spectrum clearly demonstrate the large changes in structure that occur as a function of the state of oxidation of the disulfide and at different temperatures. Thus, isolated pieces of BPTI held together by a native disulfide bond are induced to form native-like architecture. Fry et at (108) have characterized a synthetic 45-mer that corresponds in sequence to the amino terminus of adenylate kinase. This long peptide binds ATP with the same affinity as intact enzyme, and 2D-NMR studies suggest that, some fraction of the time, it assumes the correct native structure consist­ ing of two alpha helices and two beta strands ( l 08). Mutant proteins that are unable to stably enter the N state in aqueous buffer at neutral pH may also exhibit some of the properties of Do. ShortIe & Meeker

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(23) have made short deletions by removing a small number of residues from either the amino-terminal or carboxy terminal ends of staphylococcal nuclease for such model studies. For this protein and many others, the ends assume a peripheral position in the native structure, and thus this modification of the amino acid sequence may not grossly perturb most of the potential in­ teractions that occur in the D state. Analysis of the CD spectra of two large nonsense fragments of staphylococcal nuclease suggested that the wild-type Do may exhibit approximately 50% of native secondary structure. Extrapola­ tion of the gel filtration behavior of these fragments to normal chain length suggested that this state may occupy a volume only 30-50% larger than the N state (23). In addition, all of the mutants with increased values of mGuHCI exhibited significantly less residual structure , supporting the conclusion dis­ cussed above that changes in mGuHCI reflect an alteration in D state structure. SUMMARY

The denatured "state" of a protein is a distribution of many different molecu­ lar conformations, the averages of which are measured by experiments. The properties of this ensemble depend sensitively on the solution conditions. There is now considerable evidence that even in strong denaturants such as 6M GuHCI and 9M urea, some structure may remain in protein chains. Under milder or physiological conditions, the denatured states of most proteins appear to be highly compact with extensive secondary structure. Both theoret­ ical and experimental studies suggest that hydrophobic interactions, chain conformational entropies, and electrostatic forces are dominant in determin­ ing this structure. The denaturation reaction of many proteins in GuHCI or urea can be most simply modelled as a two-state transition between the native structure and a relatively compact denatured state, which then undergoes a gradual increase in radius on further addition of denaturant. However, when a protein acquires a large net charge in acids or bases, it can have two stable denatured populations, one compact and the other more highly unfolded. The prediction and elucidation of the structural details of the non-native states of proteins may ultimately prove to be as difficult as predicting the native structures, particularly for Do, the denatured state under physiological conditions. Just as with the native state, the structure of this biologically important denatured state appears to depend on the amino acid sequence. The development of synthetic peptide and protein fragment models of the de­ natured state and the recent progress in NMR spectroscopy provide bases for optimism that new insights will be gained into this poorly understood realm of protein biochemistry.

DENATURED STATES OF PROTEINS

82 1

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ACKNOWLEDGMENTS

We thank R . L. B aldwin, Linda De Young, Tony Fink, and Oleg Ptitsyn for helpful comments and K. Kuwajima, C. Dobson , C. R. Matthews, P. E. Wright, P. Kim, Elisha Haas, Fred Richards, and C. N . Pace for helpful communications . Research in the laboratories of the authors has been sup­ ported by grants from the National Institutes of Health (NIH) and the Univer­ sity Research Initiative of the Defense Advanced Research Projects Agency (DARPA) (K. A. D . ) .

APPENDIX In this appendix, we show that the observation of noncoincident sigmoidal curves of two different properties for the denaturation of a protein does not constitute proof of the existence of an intermediate state of the type shown in Figure lD . Noncoincidence of sigmoidal denaturation curves is also con­ sistent with the variable two-state model shown in Figure l B . Suppose the fraction of protein molecules in the denatured state, !D(X) has the sigmoidal dependence on denaturing agent x shown in Figure 9. Suppose one property , a, has the value aN for pure native protein and the value aD for pure denatured protein; fOT example see Figure 1 . Then the averaged value,

Denatured states of proteins.

The denatured "state" of a protein is a distribution of many different molecular conformations, the averages of which are measured by experiments. The...
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