Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.     

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.     

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.     

Equilibrium and kinetics of the thermal unfolding of alpha-lactalbumin. The relation to its folding mechanism.

The thermal unfolding of alpha-lactalbumin has been studied by equilibrium measurements of aromatic difference spectra, and by kinetic measurements of the Joule heating temperature-jump. The unfolding at neutral pH is a reversible two-state transition. The equilibrium transition curves are analyzed by the nonlinear squares method, which gives correct values of thermodynamic parameters based on the data in a wide range of temperature. The results are discussed in relation to the previous studies on the unfolding by guanidine hydrochloride or by acid. The thermally unfolded state, a partially unfolded species, is shown to be thermodynamically similar to but not identical with the acid state. The folding pathway deduced from the kinetic results is essentially consistent with the folding model of alpha-lactalbumin proposed previously. Large decreases in entropy and in heat capacity during the reversed activation suggest the packing of the folded segments by hydrophobic interactions, while the forward activation shows a marked temperature dependence, probably caused by the disruption of specific long-range interactions.     

Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.     

Transition state in the folding of alpha-lactalbumin probed by the 6-120 disulfide bond.

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.     

Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism

Kinetic refolding reactions of ferricytochrome c and beta-lactoglobulin have been studied by stopped-flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side-chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped-flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.     

Unfolding of class A amphipathic peptides on a lipid surface

The folding of polypeptides associated with biomembranes is a ubiquitous phenomenon, yet the thermodynamics underlying the process are poorly understood. In the present work we examine the unfolding of a series of alpha-helical amphipathic membrane-associated peptides using guanidine hydrochloride as a denaturant. The peptides are based on the class A amphipathic helix motif, and each contains a single tryptophan at sequence position 2, 3, 7, 12, or 14. The isothermal unfolding process was monitored by circular dichroism ellipticity at 222 nm to monitor changes in the helical structure of the peptide. Tryptophan fluorescence was used to probe the local changes in the environment about the indole fluorophore. The unfolding curves generated from the two experimental techniques for each peptide-lipid complex were non-coincidental, suggesting the presence of stable intermediate(s) in the unfolding. A three-state model could adequately account for the data and yielded parameters which were consistent with the presence of a partially folded intermediate structure which (i) is closer in Gibb's free energy to the folded state than the unfolded state and (ii) retains much of the interfacial and amphipathic character of the folded state. Denaturant-induced peptide dissociation from the peptide-lipid complexes was found to be negligible as confirmed by size exclusion chromatography. The results are compared with related thermodynamic data and discussed in terms of current models of peptide folding at membrane interfaces.     

Characterization of the unfolded state of bovine alpha-lactalbumin and comparison with unfolded states of homologous proteins

The unfolded states of three homologous proteins with a very similar fold have been investigated by heteronuclear NMR spectroscopy. Secondary structure propensities as derived from interpretation of chemical shifts and motional restrictions as evidenced by heteronuclear (15)N relaxation rates have been analyzed in the reduced unfolded states of hen lysozyme and the calcium-binding proteins bovine alpha-lactalbumin and human alpha-lactalbumin. For all three proteins, significant deviations from random-coil predictions can be identified; in addition, the unfolded states also differ from each other, despite the fact that they possess very similar structures in their native states. Deviations from random-coil motional properties are observed in the alpha- and the beta-domain in bovine alpha-lactalbumin and lysozyme, while only regions within the alpha-domain deviate in human alpha-lactalbumin. The motional restrictions and residual secondary structure are determined both by the amino acid sequence of the protein and by residual long-range interactions. Even a conservative single point mutation from I to L in a highly conserved region between the two alpha-lactalbumins results in considerable differences in the motional properties. Given the differences in oxidative folding between hen lysozyme and alpha-lactalbumin, the results obtained on the unfolded states suggest that residual long-range interactions, i.e., those between the alpha- and the beta-domain of lysozyme, may act as nucleation sites for protein folding, while this property of residual structure is replaced by the calcium-binding site between the domains in alpha-lactalbumin.     

Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme.

Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.     

Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.     

Highly nonexponential kinetics in the early-phase refolding of proteins at low temperatures

Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degrees C with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degrees C in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degrees C in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degrees C. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.     

Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein.     

Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry

Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.     

Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule

The native state (1)H, (15)N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state.     

A folding model of α-lactalbumin deduced from the three-state denaturation mechanism

It has been shown that α-lactalbumin undergoes a three-state denaturation, involving a helical intermediate state, on treatment with guanidine hydrochloride. The unfolding of the protein and the characteristics of the intermediate state are examined by means of circular dichroism, difference spectra and pH-jump measurements to investigate the temperature dependence and kinetic properties of the unfolding and refolding, the pH dependence of the transition between the intermediate and the fully unfolded states, and the effect of disulphide bond reduction on the stabilization of the intermediate.The results show that the long-range specific interactions such as specific electrostatic interactions and disulphide linkages are not important for stabilizing the intermediate, and that the transition between the intermediate and the fully unfolded states is extremely rapid (a relaxation time of less than one millisecond) and may correspond to the helix-coil transition of a polypeptide backbone. On the other hand, the activation parameters of the transition between the native and the intermediate states have suggested that the final stabilization by charge-pair interactions is preceded by hydrophobic interactions in the process of going from the intermediate to the native state.The mechanism of folding of the protein is discussed, and the folding process from the fully unfolded to the native state is apparently divided into at least three main steps: (1) the formation of incipient helical structures dictated by local interactions; (2) the packing of the helical segments accompanied with hydrophobic interactions; (3) the final stabilization by the electrostatic interactions. The relevance to the current theoretical results on protein folding is also discussed.     

Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37+/-3 A at 3 M guanidine hydrochloride. F?rster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.     

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.     

Thermodynamics of protein cross-links

The thermal transitions of native lysozyme and a well-characterized cross-linked derivative of lysozyme [Imoto, T., and Rupley, J. A. (1973), J. Mol. Biol. 80, 657] have been studied in 1.94 M guanidine hydrochloride at pH 2. The observed increase in the melting temperature from 32.4 degrees C for native lysozyme to 61.8 degrees C for the cross-linked derivative corresponds to a calculated 5.2 kcal/mol increase in the free energy of denaturation. This free-energy change is attributed to the decreased entropy of the unfolded polypeptide chain following introduction of a cross-link and is shown to compare well with theoretical predictions. The possibility that an introduction of a cross-link could also affect the enthalpy of an unfolded protein was investigated. The heats of reduction of bovine serum albumin and lysozyme by dithioerythritol in 6 M guanidine hydrochloride were determined and compared to that for the model peptide, oxidized glutathione. The near identity of the observed heats was taken as evidence that the introduction of cross-links into a random-coil protein does not, in general, introduce strain.