Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60-100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.     

Examination of the folding of E. coli CspA through tryptophan substitutions

Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism. However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated. The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site. To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein. The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing. Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants. These results indicate that the fast folding of CspA is driven by a concerted mechanism.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A hydrophobic cluster forms early in the folding of dihydrofolate reductase

The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site-directed mutagenesis and by physicochemical means. The absence of this fluorescence-detected phase in the refolding of the Trp-74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp-74. In addition, water-soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and native-like conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp-74 from solvent.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Comparison of the folding processes of T. thermophilus and E. coli ribonucleases H

In order to examine how the stabilization of thermophilic proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange. Like its homolog from Escherichia coli, this thermophilic protein populates a partially folded kinetic intermediate within the first few milliseconds of folding. The structure of this intermediate is similar to that of E.coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the protein. Proline isomerization appears to partly limit the folding of the thermophilic but not the mesophilic protein. Lastly, unlike other thermophilic proteins, which unfold much more slowly than their mesophilic counterparts, T.thermophilus RNase H folds and unfolds with overall rates similar to those of E.coli RNase H.     

Protein folding following synthesis in vitro and in vivo: association of newly synthesized protein with 50S subunit of E. coli ribosome

In the accompanying paper, it was shown that a protein, while reverting to native form from the unfolded state in vitro with the help of bacterial 70S ribosome, split the latter into its subunits (50S and 30S) and remains associated with the 50S subunit. Here, we follow the fate of nascent proteins both in case of in vivo and in vitro translation system. The newly synthesised protein was found to associate with the 50S subunit in both the cases.     

Rapid collapse precedes the fast two-state folding of the cold shock protein

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by F?rster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.     

Characterization of a slow folding reaction for the alpha subunit of tryptophan synthase

The equilibria and kinetics of urea-induced unfolding and refolding of the alpha subunit of tryptophan synthase of E. coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate-limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomalous result was shown to be due to the stabilizing effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free-energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, -47 cal/deg-mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino-acid substitutions at the interface.     

Kinetic barriers and the role of topology in protein and RNA folding

This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway.     

Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models.

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.     

Probing the interactions between the folding elements early in the folding of Escherichia coli dihydrofolate reductase by systematic sequence perturbation analysis

One of the necessary conditions for a protein to be foldable is the presence of a complete set of folding elements (FEs) that are short contiguous peptide segments distributed over an amino acid sequence. Previous studies indicated the FE assembly model of protein folding, in which the FEs interact with each other and coalesce to form an intermediate(s) early in the folding reaction. This suggests that a clue to the understanding of the determinants of protein foldability can be found by investigating how the FEs interact with each other early in the folding and thereby elucidating roles of the FEs in protein folding. To reveal the formation process of FE-FE interactions, we studied the early folding events of Escherichia coli dihydrofolate reductase (DHFR) utilizing systematic sequence perturbation analysis. Here, systematic single amino acid substitutions were introduced inside of the FEs (W30X in FE2, V40X in FE3, N59X in FE4, and I155X in FE10; X refers to various amino acid residues), and their kinetic refolding reactions were measured by stopped-flow circular dichroism and fluorescence. We show that the interactions around Trp30 and Ile155 are formed in the burst phase intermediate, while those around Val40 and Asn59 are formed in the transition state of the subsequent folding phase (tau5-phase) and in much later processes, respectively. These and previous results suggest that FE2 and FE10, and also FE1 and FE7, involved in the loop subdomain of DHFR, interact with each other within a millisecond time range, while the stable FE3-FE4 interactions are formed in the later processes. This may highlight the important roles of the FEs mainly inside of the loop subdomain in formation of the burst phase intermediate having a hydrophobic cluster and native-like overall topology and in acquisition of the foldability of DHFR.     

Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.

Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence.     

Charge centers and formation of the protein folding core

Electrostatic interactions are important for protein folding. At low resolution, the electrostatic field of the whole molecule can be described in terms of charge center(s). To study electrostatic effects, the centers of positive and negative charge were calculated for 20 small proteins of known structure, for which hydrogen exchange cores had been determined experimentally. Two observations seem to be important. First, in all 20 proteins studied 30-100% of the residues forming hydrogen exchange core(s) were clustered around the charge centers. Moreover, in each protein more than half of the core sequences are located near the centers of charge. Second, the general architecture of all-alpha proteins from the set seems to be stabilized by interactions of residues surrounding the charge centers. In most of the alpha-beta proteins, either or both of the centers are located near a pair of consecutive strands, and this is even more characteristic for alpha/Beta and all-beta structures. Consecutive strands are very probable sites of early folding events. These two points lead to the conclusion that charge centers, defined solely from the structure of the folded protein may indicate the location of a protein's hydrogen exchange/folding core. In addition, almost all the proteins contain well-conserved continuous hydrophobic sequences of three or more residues located in the vicinity of the charge centers. These hydrophobic sequences may be primary nucleation sites for protein folding. The results suggest the following scheme for the order of events in folding: local hydrophobic nucleation, electrostatic collapse of the core, global hydrophobic collapse, and slow annealing to the native state. This analysis emphasizes the importance of treating electrostatics during protein-folding simulations.     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Role of a solvent-exposed aromatic cluster in the folding of Escherichia coli CspA

Escherichia coli CspA is a member of the cold shock protein family. All cold shock proteins studied to date fold rapidly by an apparent two-state mechanism. CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998). To elucidate the role this aromatic cluster plays in the determining the folding rate and pathway of CspA, we have studied the folding kinetics of mutants containing either leucine or serine substituted for Phe 18, Phe20, and/or Phe31. The leucine substitutions are found to accelerate folding and the serine substitutions to decelerate folding. Because these residues exert effects on the free energy of the folding transition state, they may be necessary for nucleating folding. They are not responsible, however, for the very compact, native-like transition state ensemble seen in the cold shock proteins, as the refolding rates of the mutants all show a similar, weak dependence of unfolding rate on denaturant concentration. Using mutant cycle analysis, we show that there is energetic coupling among the three residues between the unfolded and transition states, suggesting that the cooperative nature of these interactions helps to determine the unfolding rate. Overall, our results suggest that separate evolutionary pressures can act simultaneously on the same group of residues to maintain function, stability, and folding rate.     

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.     

Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli

Penicillin amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an alpha-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.