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The protein FKBP52 is a steroid hormone receptor coactivator likely involved in neurodegenerative disease. A series of small, water-soluble, bioinspired, pseudopeptidic fluorescent ligands for the FK1 domain of this protein are described. The design is such that engulfing of the ligand in the pocket of this domain is accompanied by hydrogen-bonding of the dansyl chromophore which functions as both an integral part of the ligand and a fluorescent reporter. Binding is concomitant with a significant wavelength shift and an enhancement of the ligand fluorescence signal. Excitation of FK1 domain native tryptophan residues in the presence of bound ligand results in Förster resonance energy transfer. Variation of key ligand residues within the short sequence was undertaken, and the interaction of the resulting library with the protein was measured by techniques including isothermal calorimetry analysis, fluorescence, and FRET quenching, and a range of K d values were determined. Cocrystallization of a protein ligand complex at 2.30 Å resolution provided detailed information at the atomic scale, while also providing insight into native substrate binding.

The idea of a possible control of metal ion properties by constraining the coordination sphere geometry was introduced by Vallee and Williams with the concept of entasis, which is frequently postulated to be at stake in metallobiomolecules. However, the interactions controlling the geometry at metal centers remain often elusive. In this study, the coordination properties toward copper ions—Cu(II) or Cu(I)—of a geometrically constrained glycoligand centered on a sugar scaffold were compared with those of an analogous ligand built on an unconstrained alkyl chain. The sugar-centered ligand was shown to be more preorganized for Cu(II) coordination than its open-chain analogue, with an unusual additional stabilization of the Cu(I) redox state. This preference for Cu(I) was suggested to arise from geometric constraints favoring an optimized folding of the glycoligand minimizing steric repulsions. In other words, the Cu(I) d(10) species is stabilized by valence shell electron pair repulsion (VSEPR). This idea was rationalized by a theoretical noncovalent interactions (NCI) analysis. The cumulative effects of weak forces were shown to create an efficient buckle as in a hook-and-loop fastener, and fine structural features within the glycoligand reduce repulsive interactions for the Cu(I) state. This study emphasizes that monosaccharide platforms are appropriate ligand backbones for a delicate geometric control at the metal center, with a network of weak interactions within the ligand. This structuration availing in glycoligands makes them attractive for metallic entasis.

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome c (cyt c) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] of 9-10 M, which was analyzed under the framework of the equilibrium between two states (native-unfolded). In the presence of polymers, the native folding of cyt c was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.

In coordination chemistry, ligand shape can be used to tune properties, such as metal selectivity, coordination number, electronic structure, redox potential, and metal center stereochemistry including coordination helicates formation, and also to generate cavities for encapsulation. The results presented in this article indicate that two epimeric glycoligands (3 and 4) based on the conformationally restrained xylo- and ribo-1,2-O-isopropylidenefurano scaffolds are preorganized in water through pi-pi stacking due to hydrophobic interactions, as evidenced from excimer observation. The structure obtained in the solid state for one of the Cu(II) complexes (5) is chiral, with an original helical chirality arising from the coiling of the two ligands around the Cu-Cu axis. It shows an unusual double-deck type structure, with pi-pi interaction between two triazoyl-pyridyl rings and with a small cavity between the two Cu(II) ions able to host a bridging water molecule, as suggested by electron paramagnetic resonance. The Cu(II) complex from the epimeric ligand (6) shows similar properties with a mirror-image CD spectrum in the d-d region of the Cu(II). There is a predetermination of chirality at the metal center by the glycoligand induced by the C3 configuration, 6 and 5 being pseudoenantiomers. Interestingly, the stereochemistry at the metal center is here controlled by the combination of pi-stacking and chiral backbone.

A pair of novel C3-epimeric sugar-derived ligands (glycoligands) with a neutral N4O donor set was synthesized. Copper(II) complexes of both ligands were obtained and characterized by X-ray crystallography. Cyclic voltammetry, electron paramagnetic resonance, and UV-vis spectroscopies showed similar electronic properties. Mirror-image CD spectra were obtained for the Cu(II) d-d band, indicating an enantiomeric character of the coordination sphere, which has been rationalized structurally. This example shows the possible predetermination of stereochemistry for complexes by ligands based on a glycoscaffold.

Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent. Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold. We used in vitro evolution methods to demonstrate that this approach is feasible. We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted. We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin. Several different binders were selected from each library. The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail. K(D) values of about 20 nM were obtained for streptavidin-bound testosterone. The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone. The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target. Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition. Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone. These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family. This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications.

The denatured states of a small globular protein, apo-neocarzinostatin (NCS), have been characterized using several techniques. Structural properties were investigated by optical spectroscopy techniques and small-angle neutron scattering (SANS), as a function of guanidinium chloride (GdmCl) concentration. SANS experiments show that in heavy water, the protein keeps its native size at GdmCl concentrations below 2.5 M. A sharp transition occurs at about 3.6 M GdmCl, and NCS behaves like an excluded volume chain above 5 M. The same behavior is observed in deuterated buffer by fluorescence and circular dichroism measurements. For the H(2)O buffer, the transition occurs with lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe for studying the early stages of protein unfolding. Protein denaturation modifies the fluorescence intensity of ANS, a maximum of intensity being detected close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfolding pathway. Differential scanning calorimetry (DSC) was used to obtain thermodynamic values for NCS denaturation. The melting curves recorded between 20 and 90 degrees C in the presence of various GdmCl concentrations (0-3 M) cannot be explained by a simple two-state model. Altogether, the data presented in this paper suggest that before unfolding the protein explores a distribution of states which is centered around compact states at denaturant concentrations below 2 M in H(2)O, and then shifts to less structured states by increasing denaturant concentrations.

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.

Tyrosine-containing model peptides were oxidized by horseradish peroxidase (HRP). This led to a peptide polymerization via condensation of the aromatic rings. Dimers, trimers, and tetramers (depending on the peptide length and on the position of the tyrosine in the sequence) were identified by electron spray mass spectroscopy. The second-order rate constants of the second step of the HRP reduction (CII --> E) was decreased by the presence of a positively charged amino group in the vicinity of the aromatic ring as determined by stopped flow measurements [k3 = 19 398 M-1 s-1 and k3 = 1016 M-1 s-1 for N-acetyltyrosine (NAT) and l-Tyr oxidations, respectively]. High-performance liquid chromatography was used to follow the kinetics of polymerization of some model peptides after their enzymatic oxidation. The first polymerization products exhibited a strong inhibitory effect toward further oxidation by HRP. This effect was not observed when using manganese-dependent peroxidase (MnP) which does not bind directly to the tyrosine residue but rather acts as a "distant catalyst". Saturation of the HRP was achieved with Pro-Gln-Gln-Pro-Tyr (kcat = 58 s-1, = 2.1 mM), NAT (kcat = 94 s-1, = 5.6 mM), and Gly-Tyr (kcat = 175 s-1, = 10.8 mM). Analysis of steady state kinetics of the reaction showed that the dimers formed initially behaved like competitive inhibitors. The value of the dissociation constant between HRP and dimers was 20 microM. A simplified model which accounts for these observations, including the formation of a Michaelis-Menten-like complex involving the donor and enzyme, is proposed and discussed.

Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.

The Escherichia coli outer membrane ferrichrome transporter FhuA was purified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside). The amount of dodecyl maltoside bound to the protein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodecyl maltoside complex (Rs = 4.2 nm) were determined using size exclusion chromatography. Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the protein was monomeric. The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for the complex. Sedimentation experiments gave values for the molecular mass of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes radius in complete agreement with those calculated from size exclusion chromatography. The circular dichroism spectrum indicated a 51% beta-sheet content. Functionality of the purified protein was assessed from fluorescence measurements using the DNA probe YO-PRO-1. Interaction of nM concentrations of FhuA with bacteriophage T5 resulted in the release of 90 +/- 8% of the phage DNA. The limiting step in DNA ejection was binding of the phage to its receptor. Release of DNA took place in a few seconds. Ferrichrome (0.8 microM) competed with the phage for binding to FhuA and prevented DNA ejection.

A set of protein fragments was produced by site-directed mutagenesis followed by chemical cleavage of phosphoglycerate kinase according to a previously described method [Pecorari et al. (1993) Protein Eng. 6, 313-325]. The cleavage positions were chosen in order to correspond to limits between structural subdomains. These isolated fragments were studied by circular dichroism, folding transitions, and cross-linking analyses. It appears that fragments corresponding to globular subdomains in the protein can recover the expected helix content. However, the cooperativity classically observed in the folding transitions of natural proteins is only observed for fragments larger than a domain. Previous studies have shown that the isolated C-terminal domain is an autonomous folding unit which displays a single cooperatine transition [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. The results presented here show that the presence in a fragment of a sequence overpassing that of the C-terminal domain modifies its folding process. Reassociation experiments suggest that the efficiency of the complementation process is not related to the folding autonomy of the isolated fragments.

The role of domains in protein folding has been widely studied and discussed. Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme [Minard, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Protein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams, R. J. P. (1989) Eur. J. Biochem. 184, 617-625; Missiakas, D., Betton, J. M., Minard, P., & Yon, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gene were constructed. The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively. These two proteins were overexpressed and purified. They exhibit 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules. The unfolding studies reveal a large decrease in stability. From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure.

Two-dimensional 1H nuclear magnetic resonance spectroscopy is used to determine the structure of the C-terminal complementary peptide (404-415) bound to a mutant phosphoglycerate kinase (1-403). Conformational changes in the peptide induced by the formation of the peptide-protein complex are followed by transferred nuclear Overhauser effect spectroscopy. Measurement of transferred NOEs and molecular modeling reveal an alpha-helix fold in the 405-409 region. This fold is in good agreement with the corresponding helix XIV of the crystallographic structure of wild-type PGK (Watson et al., 1982). The role of the alpha-helix from the C-terminal peptide in the recovery of catalytic activity in the mutant PGK is discussed.

In order to determine the role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase, a mutant deleted of the 12 C-terminal residues (PGK delta 404-415) was constructed. This mutant folds in a conformation very similar to that of the wild-type protein, but exhibits a very low activity (0.1% of that of the wild-type enzyme). The main structural effect of the deletion of the C-terminal helix is an increase in flexibility of the whole protein and a decrease in stability by about 5 kcal/mol. The structural properties of the truncated protein are very similar, at least qualitatively, to those in the isolated domains. The accessibility of the thiol group of Cys 97 is identical to that in the isolated N-domain. The large solvent effect on the tryptophan fluorescence in the native protein at very low concentration of denaturant reveals an increase of flexibility of the C-domain, similar to that observed on the isolated C-domain. NMR measurements show that the pH dependence of His C2H and C4H chemical shifts in the truncated protein perfectly matches those of the isolated domains. The addition of the missing peptide provokes a 40-fold increase in enzyme activity at saturation. A dissociation constant of 80 microM was determined. This peptide, which displays a random structure in solution, folds in a helical structure in the region 405-410 as assessed by TRNOESY. All these results show that the C-terminal part of yeast phosphoglycerate kinase is not necessary for most of the initial folding steps but acts to lock the C-domain on the N-domain, thus ensuring the expression of full enzyme activity. Without this sequence, the protein has the sum of the properties of the two isolated domains.

The unfolding-refolding transition of phosphoglycerate kinase followed by steady-state fluorescence has clearly shown the existence of a hyperfluorescent form [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. In order to determine the contribution of each of the two tryptophans to the fluorescence properties of the enzyme in the equilibrium transition and to characterize the hyperfluorescent form, two single tryptophan mutants in which tryptophans 308 and 333 were replaced by a tyrosine and a phenylalanine, respectively, were constructed. Neither the catalytic nor the physicochemical properties of the enzyme are significantly altered by these mutations. The unfolding-refolding transitions were studied using circular dichroism and tryptophan fluorescence emission. Both tryptophans contribute to the hyperfluorescence observed in the first transition. For guanidine hydrochloride concentrations higher than 0.9 M, it clearly appears that the second transition results from a further unfolding. It is accompanied by a decrease in fluorescence intensity and a 5 mm red shift of the maximum emission wavelength. When the unfolding is induced by urea, the end of the transition corresponds to the hyperfluorescent state. Further addition of guanidine hydrochloride induces complete unfolding. These results suggest the presence of residual microstructures around tryptophan 308 and tryptophan 333 in the hyperfluorescent state. The characterization of these clusters and their contribution as starting structures in the folding process are now under investigation.

The unfolding-refolding kinetics of yeast phosphoglycerate kinase were studied using the chemical reactivity of genetically introduced cysteinyl residues as conformational probes and far-ultraviolet circular dichroism. A unique internal cysteinyl residue was introduced in several mutants at selected positions in the N- and C-domains. The cysteinyl residues were at positions 97 (the unique cysteinyl residue of the wild-type enzyme), 183 in the N-domain, 285 and 324 in the C-domain. A similar strategy has been used to study the unfolding-refolding transition under equilibrium conditions [Ballery et al. (1990) Protein Eng. 3, 199-204]. Except for the mutant C97A,A183C, whose cysteinyl residue is located at the domain interface, three labeling phases were observed during the refolding process, indicating the presence of three species, the unfolded, intermediate, and folded proteins. The comparison of the data obtained following the accessibility of the thiol group to 5,5'-dithiobis(2-nitrobenzoate) and ellipticity at 218 nm indicated that all mutants have the same folding pathway and allowed us to characterize the intermediate. In this species, each domain appeared to have a high content of secondary structure but a flexible tertiary structure; this intermediate, which had the characteristics of a molten globule, remained in fluctuating equilibrium with a widely unfolded form. The same folding intermediate was detected for mutant C97A,A183C; however, the cysteinyl residue being totally accessible to the reagent, it is likely that in this intermediate the interdomain interactions are not established. Domain pairing and formation of the native tertiary structure occur simultaneously in the slow phase of refolding. The validity and limitations of the methodology are discussed.

Equilibrium and kinetic studies of the unfolding-refolding of phage T4 lysozyme induced by guanidine hydrochloride are reported. Tryptophan fluorescence and circular dichroism (CD) were used as observables. Several results indicated the existence of intermediates in the unfolded-folded transition, including (1) the noncoincidence of the transition observed by fluorescence and CD, (2) the asymmetry of the transition recorded by CD, and (3) triphasic kinetics, followed by both observables, accounting for the forward and reverse processes. Our data were inconsistent with an independent unfolding or refolding of each domain. They indicated the existence of residual structured regions particularly resistant to denaturant, which involved one or more of the three tryptophans located in the C-terminal domain. Kinetic analysis has made it possible to propose a minimum pathway corresponding to a sequential refolding, with dead-end species arising from an intermediate. We compared our data with the experimental results of Elwell and Schellman [Elwell, M., & Schellman, J.A. (1975) Biochim. Biophys. Acta 386, 309-313] and the theoretical analysis of B. Maigret (unpublished results) and proposed that the C-terminal domain refolds first, after which the overall structure can be formed and stabilized.

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.

The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these different data and the thermodynamic analysis, it was suggested that the two domains of the horse muscle phosphoglycerate kinase refold independently of one another with different equilibrium constants, the most favorable constant referring to the folding of the C-terminal domain which contains all tryptophans.

The accessibility of peptide bonds to cleavage by Staphylococcus aureus V8 protease bound on a Sepharose matrix was used as a conformational probe in the study of the unfolding-folding transition of phosphoglycerate kinase induced by guanidine hydrochloride. It was shown that the protein is resistant to proteolysis below a denaturant concentration of 0.4 M. The transition curve, determined by susceptibility toward proteolysis, was similar to that obtained following the enzyme activity [Betton et al. (1984) Biochemistry 23, 6654-6661]. Proteolysis under conditions where the folding intermediates are more populated, i.e., 0.7 M Gdn.HCl, gave two major fragments of Mr 25K and 11K, respectively. The 25K polypeptide fragment was identified as the carboxy-terminal domain. Its conformation was similar to that of a folding intermediate trapped at a critical concentration of denaturant, and in this form, it was not able to bind nucleotide substrates [Mitraki et al. (1987) Eur. J. Biochem. 163, 29-34]. From the present data and those previously reported, we concluded that the intermediate detected on the folding pathway of phosphoglycerate kinase has a partially folded carboxy-terminal domain and an unfolded amino-terminal domain.