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The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. Nuclear magnetic resonance studies had suggested that in chicken c-CrkII a native state prolyl cis-trans isomerization is involved in signal propagation. Corresponding evidence for the closely related human c-CrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologues and found that cis-trans isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. The transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein, as well. We suggest that Pro238 isomerization extends the lifetime of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device.

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding.

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.

Often protein folding reactions show complex kinetics, because multiple unfolded species are present, which refold simultaneously. After conformational unfolding, these species are formed by the slow cis/trans equilibrations at Xaa-Pro peptide bonds. To dissect the roles of individual prolines for unfolding and refolding, we used ribonuclease T1, a protein with two cis prolyl peptide bonds, preceding Pro39 and Pro55, and two variants with substitutions at these positions. A stopped-flow double-mixing technique was employed (i) to measure the rates of the individual prolyl isomerizations in the unfolded proteins and (ii) to study the refolding of transient species that are not well populated at equilibrium. In particular, the elusive species with correct prolyl isomers could be produced by short unfolding pulses, and its refolding kinetics could be measured. The two isomerizations in unfolded ribonuclease T1 could be assigned to Pro39 and Pro55, because they occurred with almost identical rates in the wild-type protein, in the single-cis proline variants, and in tetrapeptide-4-nitroanilides, which contained prolines in the same sequential context at Pro39 and Pro55 or ribonuclease T1. The direct refolding reaction of the unfolded molecules with correct prolyl isomers shows a time constant of 180 ms (at 25 degrees C, pH 4.6). This reaction is almost unaffected by the proline substitutions. It depends nonlinearly on temperature with a maximum near 25 degrees C, which suggest that the activated state for this reaction resembles the native rather than the unfolded state in heat capacity. The formation of a transient intermediate with incorrect prolyl isomers could be studied as well. Surprisingly, this reaction is only about 5-fold slower than direct folding, and it is also accompanied by a strong decrease in the apparent heat capacity.

In the absence of its two disulfide bonds, ribonuclease T1 can exist in a native-like folded conformation when > or = 2 M NaCl is present. We measured the kinetics of unfolding and refolding of two reduced and carboxymethylated variants of ribonuclease T1 with one cis proline (the Ser54Gly/Pro55Asn variant) and with two cis prolines (the wild-type protein) as a function of the NaCl concentration. Single and double mixing techniques were used. Analysis of the kinetic results demonstrates that the two cis prolyl bonds at Pro39 and Pro55 remain cis in the folded state after the reduction and carboxymethylation of the disulfide bonds. Folded molecules with trans isomers could not be found. The substitution of cis-Pro55 influences the proline-limited folding reaction, and the analysis of the changes in the folding kinetics shows that the trans-->cis isomerizations of both prolines are slow and are rate-determining steps for the refolding of ribonuclease T1 in the presence as well as in the absence of the disulfide bonds. The direct folding reaction of protein chains with correct prolyl isomers is also affected by the Ser54Gly/Pro55Asn mutation. The rate of refolding is decreased, whereas the rate of unfolding is almost unaffected. The kinetic analysis points to two main consequences of the Ser54Gly/Pro55Asn mutation for the stability and the folding mechanism of RNase T1. It is moderately destabilizing, because the deletion of a conformationally restricted residue (Pro55-->Asn) and the insertion of a flexible residue (Ser54-->Gly) both tend to increase the entropy of the unfolded state. The cis trans isomerization of Pro55 is abolished, however, leading to a decrease in the entropy of the unfolded protein. These two entropic contributions seem to partially compensate each other, and the net change in free energy as a consequence of the Ser54Gly/Pro55Asn double mutation is very small.

We present a kinetic method to determine the concentration of native molecules in protein folding transitions. It is based on the observation that frequently native protein molecules unfold slowly when transferred to unfolding conditions, whereas folding intermediates unfold rapidly. The fraction of native molecules in a folding transition can thus be determined by kinetic unfolding assays in a two-step procedure. Aliquots of the protein are first equilibrated at different concentrations of denaturant and then transferred to constant unfolding conditions to determine the amplitude of unfolding. This amplitude is a direct measure for the concentration of native molecules in the sample. The two-state character of a solvent-induced unfolding transition can thus be examined. When the fractional change of a spectral property in a transition follows the decrease in the concentration of the native molecules, as measured by the unfolding assays, then the presence of intermediates that differ from the unfolded protein in this property can be definitely excluded. This test complements the calorimetric test for intermediates in thermal unfolding transitions. By using this method, we show that the NaCl-induced folding transition of the reduced and carboxymethylated form of a variant of ribonuclease T1 is well described by the two-state approximation. In the unfolding of apo-alpha-lactalbumin, the measured profile for the native protein coincides with the fluorescence-detected transition, but not with the transition that is monitored by amide circular dichroism. This confirms that a partially folded intermediate is present in the folding transition of apo-alpha-lactalbumin.

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.

Folding of bovine pancreatic ribonuclease A (RNase A) is a sequential process which involves the formation of well-populated structural intermediates under suitable conditions. Two intermediates have been detected on the major slow-refolding pathway of RNase A: a late intermediate (IN) which already resembles the native protein in a number of properties and a rapidly formed early intermediate (I1) which shows extensive hydrogen-bonded secondary structure. Here competition experiments between refolding and proteolytic cleavage of the peptide chain are described which yield information about the decrease in accessibility of particular proteolytic cleavage sites during the folding process. Results obtained with pepsin as a proteolytic probe of folding indicate that the primary cleavage site for pepsin, Phe-120-Asp-121, becomes inaccessible early in the course of refolding, if folding is carried out under conditions which effectively stabilize the native state. Under marginally stable conditions, folding is very slow, and protection against peptic cleavage is not detectable prior to the final formation of native protein. The comparison with amide proton exchange experiments suggests that the protection against peptic cleavage occurs during the formation and/or stabilization of hydrogen-bonded secondary structure in the early intermediate (I1). We conclude that the carboxy-terminal region of the RNase peptide chain, which is known to be important for the stability of the folded protein, may also be relevant for early steps of refolding.

Two models have been proposed to explain the observed folding kinetics of small proteins. The sequential model assumes that folding proceeds on an ordered pathway via structural folding intermediates, whereas the simple model of folding involves only multiple unfolded forms of the protein and a single native state. In the latter model, refolding is limited by interconversion reactions in the unfolded protein; accumulation of structural intermediates during folding is excluded. Here, two experimental tests are presented to discriminate between these models for the major slow folding species of ribonuclease A. The first test shows that a nativelike intermediate accumulates during folding, which unfolds rapidly compared to native ribonuclease A, and the second test demonstrates that refolding is a sequential reaction, resulting in the transient accumulation of an intermediate and in a lag in the formation of fully native protein. Both results rule out the simple model of folding and agree with the sequential model via structural intermediates. The nativelike intermediate is stable toward unfolding and is on the pathway of refolding for denaturant concentrations up to 2 M guanidine hydrochloride at pH 6 and 10 degrees C.

The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.

A transient intermediate (I3) observed previously in the unfolding of ribonuclease A has been studied by employing a sequential mixing instrument to populate selectively this species. This approach has made it possible both to determine the refolding behavior of this species and to characterize further the kinetics of its formation. (1) Formation of I3 represents the earliest detectable change in unfolding. (2) The loss of the 2'CMP binding site occurs in parallel with the exposure of the interior of the protein to solvent. (3) I3 is distinct from previously described intermediates in refolding. (4) Overall condensation of the protein to exclude solvent from the interior, as well as the formation of a substrate binding site, takes place in approximately 30 ms (pH 5.8, 47 degrees C), indicating that the formation of native structure can take place faster than had previously been supposed.