A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Molecular dynamics of the DNA-binding domain of the papillomavirus E2 transcriptional regulator uncover differential properties for DNA target accommodation.

Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA-binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA-binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the beta2-beta3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA-binding helices, and thus on the modulation of DNA recognition. A compact beta-barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose beta-barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 beta-barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable beta-barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C-terminal DNA-binding domain-DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain.

Complementation of peptide fragments of the single domain protein chymotrypsin inhibitor 2.

Chymotrypsin inhibitor 2 (CI2) folds kinetically as a single domain protein. It has been shown that elements of native secondary structure do not significantly form in fragments as the 64 residue protein is progressively increased in length from its N terminus, until at least 60 residues are present. Here, we analyse peptides of increasing length from the C terminus and find that native-like structure is not present even in the largest, fragment (7-64). We have examined sets of peptides of the form (1 - x) and ((x + 1)-64) to detect complementation. The only pair that readily complements and gives native-like structure is (1-40) and (41-64), where cleavage occurs in the protease-binding loop of CI2. But, all the pairs of peptides (1 - x) + (41-64) complement for x > 40, as do all pairs of (1-40) + (x-64), where x < 40. The resultant complexes appear to be equivalent to (1-40). (41-64) with the overlapping sequence being unstructured. Thus, the folding of CI2 is extremely co-operative, and interactions have to be made between subdomains (1-40) and (41-64). This is consistent with the mechanism proposed for the folding pathway of intact CI2 in which a diffuse nucleus is formed in the transition state between the alpha-helix in the N-terminal region of the protein and conserved hydrophobic contacts in the C-terminal region of the polypeptide. It is with these protein design features that CI2 can be an effective protease inhibitor.

Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2.

There is a region of well-ordered structure in the transition state of folding of chymotrypsin inhibitor 2 (CI2) that consists of N-terminal residues in the unique alpha-helix (residues 12 to 24) plus some long range interactions, in particular those of Ala16 with Ile57 and Leu49 in the hydrophobic core. This is proposed to be a nucleation site. A crucial question for understanding the initiation of protein folding is: when is the nucleation site formed? Is the alpha-helix pre-formed in the nominally unfolded state, or does it require long-range interactions to be stabilized? To answer this question, we have characterized a series of N-terminal fragments of CI2, each containing an increasing number of subsets of the regular secondary structure. Four small fragments have been examined by circular dichroism and two-dimensional 1H and 15N NMR spectroscopy. The smallest, [1-5], comprises the sequence corresponding to the first beta-strand of the intact protein; the second, [1-13], contains also a type III reverse turn, the second beta-strand, and a type II reverse turn; the third [1-25], consists additionally of the sequence corresponding to the alpha-helix (residues 12 to 24); the fourth, [1-28], contains, in addition, the turn following the alpha-helix. All the fragments have disordered, non-compact structure in aqueous solution. In the structure-promoting co-solvent, trifluoroethanol, alpha-helical structure is stabilized in [1-25] and [1-28] in the region corresponding to the alpha-helix in the intact protein; however, the helix is frayed at both ends and is only fractionally populated, being in dynamic equilibrium with extended conformations. These observations indicate that there is little drive for independent formation of local secondary structure in CI2, and this is reflected in the highly concerted nature of the folding reaction of this protein. The nucleation site of folding of CI2 does not accumulate in the starting state for the folding reaction, but remains embryonic until there are sufficient long range interactions to stabilize it.

Following co-operative formation of secondary and tertiary structure in a single protein module.

We have prepared a family of peptide fragments of the 64 amino acid protein chymotrypsin inhibitor (CI2), corresponding to progressive elongation from the N terminus, in order to elucidate the basis of conformational preferences in single-domain proteins and to obtain insights into their conformational pathway. Structural analysis of the fragment comprising the first 50 residues, CI2(1-50), indicates that it is mainly disordered, with patches of hydrophobic residues exposed to the solvent. Structural characterisation of the fragment CI2(1-63) which lacks only the C-terminal glycine, Gly64, shows native-like structure in all regions of the fragment. The study provides insights into the contribution of specific residues to the stability and co-operativity of the intact protein. We define a phiNMR value, derived from chemical shift analysis, which describes the build-up of structure at the level of individual residues (protons). All the macroscopic probes used to study the growth of structure in CI2 on elongation of the chain (circular dichroism, fluorescence and gel filtration) are in agreement with the residue-by-residue description by NMR. It is seen that secondary and tertiary structure build up in parallel in the fragments and show similar structures to those developed in the transition state for folding of the intact protein.

Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway.

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (C12) in order to study the development of structure on elongation of the polypeptide in solution. We present an extensive biophysical characterization of ten fragments using different conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (1-40) has non-native local hydrophobic clusters, but nevertheless does not bind 8-anilinonaphthalene-1-sulphonate (ANS). Hydrophobic regions in longer fragments become gradually more capable of binding ANS as the chain grows to completion, with a tendency to form native structures. Major changes in secondary structure and accessibility to hydrophobic sites occur in parallel, between (1-40) and (1-53), together with changes in hydrodynamic volume and flexibility. NMR studies of (1-53), the first fragment displaying tertiary interactions, show that a subcore is fully formed and the alpha-helix (residues 12 to 24) is of fluctuating structure. Fragments (1-53) and (1-60) share many properties with molten globule-like structures, with varying degrees or order. Fluorescence properties of the native fold are gradually recovered from fragments (1-60) to full-length C12, together with a decrease in hydrophobic exposure. A small degree of co-operativity of formation of structure appears when residue 60 is added, gradually increasing as residue 62 is added, but a full two-state co-operative transition appears only on addition of Arg62 and Val63. We believe this is the result of correct side-chain packing of the hydrophobic core, capping the major elements of secondary structure in C12 at this late stage, which is probed by the complete recovery of the fluorescence of the unique Trp5. The structures that develop as the polypeptide chain increases in length parallel the structural features present in the nucleus for the folding of intact protein, which develops in the transition state. The folding nucleus consists of much of the helix and the interactions made by Ala16 in the helix with residues in the core, especially with Leu49 and Ile57, with the rest of the structure being formed only very weakly in the transition state.

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.

Folding of a dimeric beta-barrel: residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain.

The dimeric beta-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV-16 domain, and, as part of these studies, we present a structural characterization of the urea-denatured state of the protein. We have obtained a set of chemical shift assignments for the C-terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3'J(NHN alpha) coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second beta-strand in the folded conformation. Most of the structures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate-limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.

Modification of the amino acid specificity of tyrosyl-tRNA synthetase by protein engineering.

The amino acid specificity of Bacillus stearothermophilus tyrosyl-tRNA synthetase was studied by site-directed mutagenesis of residues close to the active site. X-ray crystallographic studies of the enzyme have suggested that Asp-176 is a major determinant of amino acid specificity, as its carboxylate is observed to make a hydrogen bond with the hydroxyl group of the substrate tyrosine. Previous efforts to test the importance of Asp-176 by site-directed mutagenesis led to inactive enzymes. We have now investigated the catalytic properties of enzymes altered, not at Asp-176 itself, but instead at two amino acids, Asn-123 and Trp-126, that appear in the crystallographic structure to form hydrogen bonds with Asp-176. Mutation of Trp-126 does not affect the kinetics of activation with respect to ATP but leads to modest increases in the Km for tyrosine. Conversely, position Asn-123 mutants are strongly affected: 160-fold lower kcat and 5-fold higher Km for the Ala-123; and 17-fold decrease and 270-fold increase, respectively, of the same parameters for the Asp-123 mutation. The specificity against phenylalanine was determined from the ratios of kcat/Km for the amino acids in the pyrophosphate exchange reaction. The ratio of 1.2 x 10(5) for the wild-type enzyme decreases 4-fold on mutation of Asn-123 but increases 7-fold on the mutation of Trp-126-->Phe and 2-fold on Trp-126-->Leu. The wild-type enzyme has not reached the maximum limit of discrimination between tyrosine and phenylalanine.

Conformational preferences of a peptide corresponding to the major antigenic determinant of foot-and-mouth disease virus: implications for peptide-vaccine approaches.

The conformational preferences in solution of a peptide corresponding to the GH loop of the VP1 capsid protein from the foot-and-mouth disease virus were examined by proton nuclear magnetic resonance and circular dichroism. The GH loop is the major antigenic determinant of the virus and participates in cell attachment through an integrin-like Arg-Gly-Asp sequence. The synthetic peptide, corresponding to residues Gly132 to Ser162 of the VP1 capsid protein of the serotype O, is largely disordered in aqueous solution as shown by the absence of long- and medium-range NOE contacts and by random-like chemical shifts values. Helical contents in aqueous solution were estimated to be less than 10%, as determined by extrapolation of trifluoroethanol titration from CD measurements, in good agreement with estimations from NMR experiments. In the presence of 40% trifluoroethanol an alpha-helix, flanked by two proline residues between Asn12 (Asn143 in the intact protein) and Leu28 (159), is induced. This contrasts with the 3(10) helix observed between residues Leu148 and Val155 in the crystal structure of the dithiothreitol-reduced virus, indicating that the cosolvent does not stabilize a residual, low-populated structure, similar to that in the intact virus. Several algorithms also fail to predict the structure found in the intact virus because these are based mainly on local sequence information. The lack of structure of the peptide in aqueous solution strongly suggests that the conformational determinants sufficient for the structure stabilization of this highly immunogenic antigen are mostly dictated by interactions of the loop with other regions of the virus structure, and do not arise from local amino acid sequence information. The ability of designed GH-VP1 peptides to neutralize anti-virus antibodies is likely to arise from antibody-induced conformation of the peptide and its application as peptide vaccines is not straightforward. Similarly, insertion of these peptides in carriers or macromolecular assemblies as vaccine vectors would depend on the conformation adopted at the insertion site and its success cannot be predicted.

Spectroscopic characterization of the growing polypeptide chain of the barley chymotrypsin inhibitor-2.

A series of N-terminal fragments of increasing size covering the complete sequence of chymotrypsin inhibitor-2 were previously produced using a combination of peptide synthesis and chemical cleavage at engineered methionine residues. Circular dichroism (CD) and NMR spectroscopic studies of the fragments are presented, as well as the folding characterization of the methionine mutants. The far-uv circular dichroism spectra of the small fragments correspond to disordered structures with some weak interactions indicated by temperature effects on certain spectral bands. There is evidence of a formation of a beta-turn around residues 24-26 in fragment CI-2(1-28), not observable on the NMR time scale. The intermediate fragment (1-50) is disordered at low temperature but more ordered when the temperature is increased. The 1D NMR spectra of the large fragments show an increased chemical shift dispersion in the amide hydrogen region, which indicates that tertiary interactions appear in fragment (1-53) together with a major secondary structure change, as shown by the concomitant change in the CD spectra. A key structural transition occurs on addition of residues Phe-50 and Val-51 and fragment (1-60) is largely folded. A second important structural transition in the elongation of the polypeptide occurs on addition of residue Val-63, where a compact fully folded fragment (1-63) is formed albeit with largely reduced stability. Near-uv circular dichroism indicates that the environment of the Trp-5 is fully recovered only in (1-63), probing the correct side-chain packing.

Association of complementary fragments and the elucidation of protein folding pathways.

Natural or engineered sites for chemical cleavage can be used to generate complementary fragments of well characterized proteins. The peptide fragments represent a unique tool for studying early events in protein folding, since these are usually the most inaccessible to the experimentalist. Analysis of the isolated fragments in non-denaturing conditions together with the determination of the structure of the folded non-covalent complexes and, most importantly, the kinetic analysis of the resulting second-order association/folding reaction can give a more complete picture of a folding pathway. The contribution of fragments to the understanding of some well characterized protein folding pathways is discussed.

The dimeric DNA binding domain of the human papillomavirus E2 protein folds through a monomeric intermediate which cannot be native-like.

The dimeric DNA binding domain of the human papillomavirus E2 protein displays a two-state concerted unfolding and dissociation, with no detectable monomeric intermediate species accumulated at equilibrium. We investigated the kinetic folding mechanism of the dimeric domain using stopped-flow spectroscopic techniques and observed a fast forming monomeric intermediate, followed by a slower bimolecular reaction. Both phases involve secondary structure rearrangements of similar magnitude. Our results support a folding pathway in which the formation of an early monomeric intermediate, with characteristics of hydrophobic collapse, is followed by a bimolecular step encompassing association and folding. The interwoven folding topology of this particular type of dimeric beta-barrel found in the E2 DNA binding domain strongly suggests that any monomeric species formed could not be native-like.

Characterization of a partially folded monomer of the DNA-binding domain of human papillomavirus E2 protein obtained at high pressure.

The pressure-induced dissociation of the dimeric DNA binding domain of the E2 protein of human papillomavirus (E2-DBD) is a reversible process with a Kd of 5.6 x 10(-8) M at pH 5.5. The complete exposure of the intersubunit tryptophans to water, together with the concentration dependence of the pressure effect, is indicative of dissociation. Dissociation is accompanied by a decrease in volume of 76 ml/mol, which corresponds to an estimated increase in solvent-exposed area of 2775 A2. There is a decrease in fluorescence polarization of tryptophan overlapping the red shift of fluorescence emission, supporting the idea that dissociation of E2-DBD occurs in parallel with major changes in the tertiary structure. The dimer binds bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding by about 30%, in contrast with the almost complete loss of dye binding in the urea-unfolded state. These results strongly suggest the persistence of substantial residual structure in the high pressure state. Further unfolding of the high pressure state was produced by low concentrations of urea, as evidenced by the complete loss of bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded state is also apparent from the distribution of excited state lifetimes of tryptophan. The combined data show that the tryptophans of the protein in the pressure-dissociated state are exposed long enough to undergo solvent relaxation, but the persistence of structure is evident from the observed internal quenching, which is absent in the completely unfolded state. The average rotational relaxation time (derived from polarization and lifetime data) of the pressure-induced monomer is shorter than the urea-denatured state, suggesting that the species obtained under pressure are more compact than that unfolded by urea.

Conformational changes and stabilization induced by ligand binding in the DNA-binding domain of the E2 protein from human papillomavirus.

We are investigating the folding of the 81-residue recombinant dimeric DNA binding domain of the E2 protein from human papillomavirus and how it is coupled to the binding of its DNA ligand. Modifications in buffer composition, such as ionic strength and phosphate, cause an approximately 5.0 kcal mol-1 stabilization of the domain to urea unfolding, based on very similar conformational changes as measured by far UV circular dichroism. Binding of DNA produces an even greater stabilization, magnitude similar to that caused by the nonspecific polymer ligand heparin, which shifts the urea midpoint 2.5-fold. The DNA-bound complex displays substantial changes similar to those caused by ionic strength and phosphate in terms of overall secondary structure. Bis-8-anilino-1-naphthalenesulfonate provides a very sensitive conformational probe, which shows alterations in the domain caused by the above mentioned compounds. In general terms, binding of DNA involves an overall conformational readjustment in the protein but maintains the beta-barrel scaffold intact. This conformational plasticity seems to be of importance in the regulatory functions of this type of DNA-binding protein. The extremely long half-life of the E2-DNA complex, together with its very high stability, suggests that, in the absence of other factors that may affect its stability in vivo, the possibility of dissociation once formed is restricted.

Generation of a family of protein fragments for structure-folding studies. 1. Folding complementation of two fragments of chymotrypsin inhibitor-2 formed by cleavage at its unique methionine residue.

The suitability of the barley chymotrypsin inhibitor-2 for study by fragmentation and complementation has been analyzed. The primary residue for binding to proteases, Met-59 (the unique methionine in the sequence), lies in a broad, solvent-exposed loop. The bond between Met-59 and Glu-60 was cleaved by cyanogen bromide. The two fragments thus obtained, i.e., CI-2(20-59) and CI-2(60-83), associate (KD = 42 nM) to yield a complex that has fluorescence and circular dichroism spectra identical to those of uncleaved chymotrypsin inhibitor-2. Recovery of native-like structure is further indicated by the ability of the complex to inhibit chymotrypsin, although the [I]50% is 140-fold higher than for the uncleaved inhibitor. CI-2(60-83) appears to be highly disordered in water, but fragment CI(20-59) forms significative structure, as judged by its circular dichroism spectra and evidence from one-dimensional NMR. The circular dichroism spectra of CI-2(20-59) approach the baseline in 4 M guanidinium chloride but display characteristics of an alpha-helix in the presence of trifluoroethanol. Analytical ultracentrifugation shows no concentration-dependent change in the molecular weight of the monomer of CI-2(20-59). Both one- and two-dimensional NMR of the complex [CI-2(20-59).(60-83)] show unequivocally the presence of a folded structure, which appears to be slightly different from the uncleaved native protein.

Three-dimensional solution structure and stability of thioredoxin m from spinach.

Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms.

Generation of a family of protein fragments for structure-folding studies. 2. Kinetics of association of the two chymotrypsin inhibitor-2 fragments.

The kinetics of association of the fragments of the barely chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), to form a native-like structure follows two phases. There is a major second-order component with rate constant (3.7 +/- 0.3) x 10(3) M-1 s-1 and a slow first-order phase of rate constant 0.011 +/- 0.001 s-1. The major phase contains a cooperative folding process as judged by the secondary structure recovery in parallel with the fluorescence change. The time course for structure formation has uniform changes at all of the wavelengths of the circular dichroism spectra, suggesting that all elements of secondary structure are formed simultaneously. A series of kinetic experiments suggest that the association and folding occur in the second-order step and that the first-order step probably results from a cis-trans peptidylprolyl isomerization in the fragment CI-2(20-59). This was confirmed by experiments on fragments derived from two mutants whose parent proteins fold more slowly than wild-type CI-2. Those fragments display lower second-order rate constants, but the rate constants of the first-order phase are the same as for wild type. The experiments suggest that the mechanism of the association/folding of mutant fragments may be studied by a protein-engineering analysis.

The structure of the transition state for the association of two fragments of the barley chymotrypsin inhibitor 2 to generate native-like protein: implications for mechanisms of protein folding.

Possible early events in protein folding may be studied by dissecting proteins into complementary fragments. Two fragments of chymotrypsin inhibitor 2 [CI2-(20-59) and CI2-(60-83)] associate to form a native-like structure in a second-order reaction that combines collision and rearrangement. The transition state of the reaction, analyzed by the protein engineering method on 17 mutants, is remarkably similar to that for the folding of the intact protein--a structure that resembles an expanded version of the folded structure with most interactions significantly weakened. The exception is that the N-terminal region of the single alpha-helix (the N-capping box) is completely formed in the transition state for association of the fragments, whereas it is reasonably well formed for the intact protein. Preliminary evidence on the structures of the individual fragments indicates that both are mainly nonnative, lacking native secondary structure and having regions of nonnative buried hydrophobic clusters. The association reaction does not result from the collision of a subpopulation of two fully native-like fragments but involves a considerable rearrangement of structure.

Folding of a pressure-denatured model protein.

The noncovalent complex formed by the association of two fragments of chymotrypsin inhibitor-2 is reversibly denatured by pressure in the absence of chemical denaturants. On pressure release, the complex returned to its original conformation through a biphasic reaction, with first-order rate constants of 0.012 and 0.002 s-1, respectively. The slowest phase arises from an interconversion of the pressure-denatured state, as revealed by double pressure-jump experiments. Below 5 microM, the process was concentration dependent with a second-order rate constant of 1,700 s-1 M-1. Fragment association at atmospheric pressure showed a similar break in the order of the reaction above 5 microM, but both first- and second-order folding/association rates are 2.5 times faster than those for the refolding of the pressure-denatured state. Although the folding rates of the intact protein and the association of the fragments displayed nonlinear Eyring behavior for the temperature dependence, refolding of the pressure-denatured complex showed a linear response. The negligible heat capacity of activation reflects a balance of minimal change in the burial of residues from the pressure-denatured state to the transition state. If we add the higher energy barrier in the refolding of the pressure-denatured state, the rate differences must lie in the structure of this state, which has to undergo a structural rearrangement. This clearly differs from the conformational flexibility of the isolated fragments or the largely unfolded denatured state of the intact protein in acid and provides insight into denatured states of proteins under folding conditions.