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Summary Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

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.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.

SlyD is a bacterial two-domain protein that functions as a molecular chaperone, a prolyl cis/trans isomerase, and a nickel-binding protein. This review summarizes recent findings about the molecular enzyme mechanism of SlyD. The chaperone function located in one domain of SlyD is involved in twin-arginine translocation and increases the catalytic efficiency of the prolyl cis/trans isomerase domain in protein folding by two orders of magnitude. The C-terminal tail of SlyD binds Ni2+ ions and supplies them for the maturation of [NiFe] hydrogenases. A combined biochemical and biophysical analysis revealed the molecular basis of the delicate interplay of the different domains of SlyD for optimal function.

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.

Cyclophilins are ubiquitous cis-trans-prolyl isomerases (PPIases) found in all kingdoms of life. Here, we identify a novel family of cyclophilins, termed AquaCyps, which specifically occurs in marine Alphaproteobacteria, but not in related terrestric species. In addition to a canonical PPIase domain, AquaCyps contain large extensions and insertions. The crystal structures of two representatives from Hirschia baltica, AquaCyp293 and AquaCyp300, reveal the formation of a compact domain, the NIC domain, by the N- and C-terminal extensions together with a central insertion. The NIC domain adopts a novel mixed alpha-helical, beta-sheet fold that is linked to the cyclophilin domain via a conserved disulfide bond. In its overall fold, AquaCyp293 resembles AquaCyp300, but the two proteins utilize distinct sets of active site residues, consistent with differences in their PPIase catalytic properties. While AquaCyp293 is a highly active general PPIase, AquaCyp300 is specific for hydrophobic substrate peptides and exhibits lower overall activity.

Folding enzymes often use distinct domains for the binding of substrate proteins (“chaperone domains”) and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.

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.

The cis / trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.

PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264-357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.

Summary SlyD (sensitive to lysis D) is a putative folding helper from the bacterial cytosol and harbors prolyl isomerase and chaperone activities. We determined the solution NMR structure of a truncated version of SlyD (1–165) from Escherichia coli (SlyD⁎) that lacks the presumably unstructured C-terminal tail. SlyD⁎ consists of two well-separated domains: the FKBP domain, which harbors the prolyl isomerase activity, and the insert-in-flap (IF) domain, which harbors the chaperone activity. The IF domain is inserted into a loop of the FKBP domain near the prolyl isomerase active site. The NMR structure of SlyD⁎ showed no distinct orientation of the two domains relative to each other. In the FKBP domain, Tyr68 points into the active site, which might explain the lowered intrinsic prolyl isomerase activity and the much lower FK506 binding affinity of the protein compared with archetype human FKBP12 (human FK506 binding protein with 12 kDa). The thermodynamics and kinetics of substrate binding by SlyD⁎ were quantified by fluorescence resonance energy transfer. NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD⁎ as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD⁎ binding to denatured insulin. The capacity of the IF domain to establish an initial encounter–collision complex, together with the flexible orientation of the two interacting domains, makes SlyD⁎ a very powerful catalyst of protein folding.

To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula Abz-Ala-Xaa-Pro-Phe-pNA, the position Xaa before the proline is occupied by all 20 proteinogenic amino acids. A conformational analysis of the peptide by molecular dynamics simulations and byNMRspectroscopy showed that the mutual distance between the Abz and pNA moieties in the peptides depends on the isomeric state of the Xaa-Pro bond. In the cis, but not in the trans form, there are significant chemical shift changes of the Abz and pNA moieties, because their aromatic rings are close to each other. This proximity also leads to a strong quenching of Abz fluorescence, which, in combination with a solvent jump, was used to devise a sensitive assay for prolyl isomerases. Unlike the traditional assay, it is not coupled with peptide proteolysis and thus can be employed for protease-sensitive prolyl isomerases as well. The peptide library was used to provide a complete set of P1-site specificities for prototypic human members of the three prolyl isomerase families, FKBP12, cyclophilin 18, and parvulin 14. In a second application, the substrate specificity of SlyD, a protease-sensitive prolyl isomerase from Escherichia coli, was characterized and compared with that of human FKBP12 as well as with homologues from other bacteria. © 2009 American Chemical Society.

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.

The signal adapter protein c-CrkII from chicken but not from human uses isomerization at Pro238 in the SH3C domain to regulate the activity of the SH3N domain. The different behavior of human and chicken c-CrkII originates from only two differences in sequence, at positions 239 after Pro238 and 272 in the N-Src loop of SH3C. We analyzed the kinetics of substrate binding to SH3N and an assay for its coupling with Pro238 isomerization in SH3C to identify the molecular path from Pro238 to the substrate binding site of SH3N. The trans→cis isomerization at Pro238 and a relocation of Phe239 re-organize the energetics of a hydrophobic cluster in the N-Src loop of SH3C and re-shape this region to optimize its interactions with SH3N. Concomitantly, the backbone becomes strained at Met272. We suggest that, in human c-CrkII, movement at position 239 and strain at position 272 are not tolerated because the β-branched residues Ile239 and Val272 restrain the backbone mobility and thus destabilize the cis Pro238 form.

Improving the stability of proteins is a major aim in basic and applied protein science. Querol and coworkers calculated changes in the quasi-electric dipole moment of a protein and used it as a simple criterion to predict stabilizing charge mutations. They employed this method to propose for the bacterial cold shock protein Bc-Csp a number of charge mutations that should have a strong influence on stability. We produced eight variants of Bc-Csp with such mutations and measured their stabilities experimentally. However, we could not find a correlation between the stability and the quasi dipole moment of these variants. Possibly, the quasi dipole moment reflects only a secondary aspect of the changes that are caused by charge mutations in a protein.

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.

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.

Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions. Here we report that a proline is used as a molecular timer in the infection of Escherichia coli cells by the filamentous phage fd. The phage is activated for infection by the disassembly of the two N-terminal domains, N1 and N2, of its gene-3-protein, which is located at the phage tip. Pro213, in the hinge between N1 and N2, sets a timer for the infective state. The timer is switched on by cis-to-trans and switched off by the unusually slow trans-to-cis isomerization of the Gln212-Pro213 peptide bond. The switching rate and thus the infectivity of the phage are determined by the local sequence around Pro213, and can be tuned by mutagenesis.

Unfolded ribonuclease A consists of a mixture of fast-folding (UF) and slow-folding species (US); US folds slowly because it contains non-native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence-detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2′CMP binding. Under these conditions the fluorescence-detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of Us by a two-step assay for non-native proline isomers. The following conclusions are drawn. (1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non-native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence-detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect prolinc isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.

Unfolded ribonuclease A (RNase A) consists of a mixture of fast refolding (UF) and slow-refolding (US) species. The slow UF⇌US equilibration reaction is rate-limited by proline peptide bond isomerization. Investigations of the dependence on temperature of the UF⇌US equilibrium have led to conflicting results and different molecular interpretations. Here the dependence on temperature of the UF: US ratio was reinvestigated by using a new assay for the fast-folding molecules UF. Between 0°C and 60°C the proportion of UF present in unfolded RNase A at 6M guanidine · HCl was found to be independent of temperature. Consequently, no conclusions can be drawn regarding the role and importance of particular prolines in the UF⇌US transition solely from these results.

The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.

Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on a single extracellular foldase, the PrsA protein. PrsA is a 30-kDa protein, lipid anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to the trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA. Background: PrsA is a foldase for secreted proteins and pathogenicity factors in Gram-positive bacteria. Results: Crystallographic, enzymatic, and NMR spectroscopic analysis provide insights to PrsA function. Conclusion: Substrate peptides interact around a unique crevice generated by PrsA dimerization via its chaperone-like domain. Significance: The comprehensive characterization of PrsA promotes its utilization as foldase and drug target.

c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.

Background: Prolyl cis/trans isomerizations have long been known as critical and rate-limiting steps in protein folding. Results: Now it is clear that they are also used as slow conformational switches and molecular timers in the regulation of protein activity. Here we describe several such proline switches and how they are regulated. Conclusions and general significance: Prolyl isomerizations can function as attenuators and provide allosteric systems with a molecular memory. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.

The signaling protein CrkII switches between forms with high or low binding affinity. Both phosphorylation and native-state prolyl isomerization were suggested to regulate the transition between these forms. Here we analyzed how phosphorylation at Tyr222 and Tyr252 and the Pro238Ala substitution affect signal transfer of human and chicken CrkII to a downstream target. Human CrkII is regulated by phosphorylation only, but chicken CrkII is regulated by Pro238 trans→cis isomerization and by Tyr222 phosphorylation. Surprisingly, they act in an independent fashion. Apparently, the allosteric transition to a low-activity form can be induced by phosphorylation or prolyl isomerization located at distant sites in CrkII.

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.

Spinach starch debranching enzyme, a limit dextrinase or pullulanase (EC 3.2.1.41), is a monomeric protein of 100 kDa that produces up to seven coexisting and mutually interconvertible isomers of different specific activity, a phenomenon that has been termed microheterogeneity and for which a structural explanation has not yet been presented. The enzyme can be activated by reduction, in particular by thiol reagents, and inactivated by oxidation and the concomitant change of the patterns of its isomeric forms could be quantified by chromatofocusing. The hypothesis was examined that reduction of the enzyme's thiol groups shifts the isomer pattern towards the forms with a higher specific activity while oxidation favours the less active forms. Using TCEP as reductant only the form with the highest specific activity was obtained. This form was almost inaccessible for proteolysis by trypsin while the oxidized and GSH-activated enzyme yielded four peptides when treated with trypsin. Their sequence indicated cleavage predominantly of loops connecting the beta-strands and alpha-helices of the (beta/alpha)(8)-barrel which forms the catalytic site of the pullulanase. Formation of various disulphide bridges between the loops connecting the barrel structures -- predominantly on one side -- may be the reason for the microheterogeneity of the spinach pullulanase. In vivo, the enzyme maintains its activated state due to the high concentration of GSH in the chloroplast. However, the chloroplast's pH shifts from day (pH 8) to night (pH 7) and thus could also alter the activity of the protein in accordance with the required function in starch metabolism.

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol−1 at 70 °C). 11.5 kJ mol−1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

There has been some debate as to whether protein folding involves diffusive chain motions and thus depends on solvent viscosity. The interpretation of folding kinetics in viscous solvents has remained difficult and controversial, in that viscogenic agents affect folding rates not only by increasing solvent viscosity but also by increasing protein stability. By carefully choosing experimental conditions, we can now eliminate the effect on stability and show that the folding dynamics of the cold shock protein CspB are viscosity dependent. Thus Kramers' theory of reaction rates rather than transition state theory should be used to describe this folding reaction.

Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar beta-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N U two-state reaction with average lifetimes of only 100-150 ms for the native state and 1-6 ms for the unfolded states at 25 degreesC. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 degreesC as well as after cold shock. The cellular half-life of CspC is shortened at 37 degreesC, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 degreesC and, in particular, under cold shock conditions.

The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions. Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium. The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors. We used this inhibition to characterize the substrate binding sites of the trigger factors. Unfolded α-lactalbumin binds very tightly and inhibits the trigger factor from M. genitalium with a KI value of 50 nM. The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues. The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding. The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already. They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain.

In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing.

Prolyl isomerases accelerate the cis trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.

Recently a new family of prolyl isomerases was discovered, which is unrelated with the cyclophilins or the FK-506 binding proteins. Parvulin, the smallest member of this new family, is a protein with only 92 residues, but parvulin-like domains occur in several large proteins that are apparently involved in protein folding or activation processes. We show here that, in addition to its activity in assays with proline-containing tetrapeptides, parvulin catalyzes the proline-limited folding of a variant of ribonuclease T1 with a kcat/Km value of 30,000 M-1 s-1. This value is much smaller than the kcat/Km value of 1.1x10(7) M-1 s-1 determined for the parvulin-catalyzed prolyl isomerization in the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide. Parvulin itself unfolds and refolds reversibly in a simple two-state reaction with a Gibbs free energy of stabilization of 28 kJ/mol at 10 degrees C. Most of the unfolded parvulin molecules refold in a slow reaction that involves prolyl isomerization and is catalyzed by cyclophilin, another prolyl isomerase. Moreover, parvulin accelerates its own refolding in an autocatalytic fashion, and the rate of refolding increases tenfold when the concentration of parvulin is increased from 0.5 to 3.0 microM. Apparently, small single-domain prolyl isomerases catalyze prolyl isomerization much better in short peptides than in protein folding reactions, presumably because the prolyl bonds are less accessible in refolding protein chains. It is possible that the additional domains of the large prolyl isomerases improve the affinity for protein substrates.

Mia40 catalyses the oxidative folding of disulphide-containing proteins in the mitochondria. The folding pathway is directed by the formation of the first mixed disulphide between Mia40 and its substrate. Here, we employ Cox17 to elucidate the molecular determinants of this reaction. Mia40 engages initially in a dynamic non-covalent enzyme–substrate complex that forms and dissociates within milliseconds. Cys36 of Cox17 forms the mixed disulphide in an extremely rapid reaction that is limited by the preceding complex formation with Mia40. Cys36 reacts much faster than the three other cysteines of Cox17, because it neighbours three hydrophobic residues. Mia40 binds preferentially to hydrophobic regions and the dynamic nature of the non-covalent complex allows rapid reorientation for an optimal positioning of the reactive cysteine. Mia40 thus uses the unique proximity between its substrate-binding site and the catalytic disulphide to select a particular cysteine for forming the critical initial mixed disulphide. Proteins containing multiple disulphide bonds fold more efficiently if these bonds are formed in a defined order. Koch et al.find that the mitochondrial thiol oxidase Mia40 achieves this by preferentially targeting cysteine residues within a hydrophobic context.

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.

Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here, we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis → trans isomerization in the SlyD-bound state shows a rate near 100 s(-1) and is thus more than 10(4)-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of ≥1 μM SlyD, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.

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.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.

The (βα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (βα)(8)-barrel proteins have evolved from an ancestral (βα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (βα)(8)-barrel proteins were constructed by the duplication of a (βα)(4) element of a modern (βα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (βα)(8)-barrels show the same refolding mechanism as HisF and other modern (βα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (βα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.

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.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.

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.