The folding of hirudin adopts a mechanism of trial and error.

Reduced and denatured hirudin (65 amino acids and 3 disulfides) refolds in vitro to become an active molecule. The folding process adopts a mechanism of "trial and error" without predominant pathways. Throughout the entire folding process, the 6 cysteines were about equally involved in the disulfide shuffling. Among the first 20% of 3-disulfide species accumulated during the early phase of refolding, two-thirds were inactive and were reshuffled in the presence of thiol catalyst to regain correct disulfide pairing. When refolding was performed in the presence of strong denaturant (guanidinium chloride) without thiol catalyst, 8% of the active hirudin was obtained. This figure is close to the probability (6.7%) that would be expected from the random disulfide pairing of a molecule containing 6 sulfhydryl groups.     

Oxidative folding of murine prion mPrP(23-231).

A systematic study of the oxidative folding of murine prion protein mPrP(23-231) is reported here. Folding of mPrP(23-231) involves formation of a single disulfide bond, Cys179-Cys214. Despite this simplicity, reduced mPrP(23-231) exhibits numerous unusual folding properties. In the absence of denaturant, folding of mPrP(23-231) is extremely sluggish, regardless of pH. The optimal pH for mPrP(23-231) folding was found to be 4-5. At pH 8.0, a condition that typically favors disulfide formation, folding of mPrP(23-231) hardly occurs, and it not facilitated by inclusion of redox agent. In the presence of denaturant (4 M urea or 2 M guanidine hydrochloride) and basic pH (8.0), reduced mPrP(23-231) refolds to the native structure quantitatively. The efficiency of folding can be further promoted by the presence of oxidized glutathione. At pH 4.0 and in the presence of 4 M urea, reduced mPrP(23-231) converts to three distinctive conformational isomers, unable to form the native structure. These unusual properties lead us to the following conclusions. The reduced mPrP(23-231) adopts a highly rigid structure with the two cysteines buried or situated apart. The presence of denaturant or low pH disrupts this rigid structure and lowers the energy barrier, which permits oxidation and refolding of the reduced mPrP(23-231). Under selected conditions, reduced mPrP(23-231) is capable of taking on multiple forms of stable conformational isomer that are segregated by energy barriers.     

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: trimer dissociation can be separated from unfolding

The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

Kinetics of folding and unfolding of beta beta-tropomyosin.

The kinetics of folding from random coils to two-chain coiled coils of beta beta-tropomyosin was studied by stopped-flow CD (SFCD) in the backbone region (222 nm). Two species were studied: the reduced form and the doubly disulfide cross-linked form. The proteins were totally unfolded in 6M urea-saline buffer, then refolded by tenfold dilution into benign buffer. In the refolding medium, they spontaneously recover the two-chain coiled-coil structure. Reduced beta beta refolds in at least two stages: one or more fast phases (< 0.04 s), in which an intermediate with 71% of the equilibrium ellipticity forms, followed by a slower time-resolvable phase that completes the folding. The slow phase is first order, signifying that dimerization occurs in the fast phase. The time constant of the slow phase is 2 s at 20 degrees C and requires activation parameters of delta S not equal to = -7 +/- 0.3 cal/mol.K, delta H not equal to = 15 +/- 1 kcal/mol. These results are very similar to those previously found for the reduced genetic variant alpha alpha-tropomyosin. In contrast, refolding of doubly disulfide cross-linked beta beta is complete within the dead time (< 0.04 s), whereas the singly cross-linked alpha alpha species also displays a slow phase. The opposite process, unfolding reduced beta beta from the coiled-coil state, is complete within the dead time, as in the alpha alpha variant.     

Intermediates in the refolding of ribonuclease at subzero temperatures. 3. Multiple folding pathways

The kinetics of refolding of ribonuclease A have been measured at -15 degrees C by monitoring the intrinsic fluorescence and absorbance signals from the six tyrosine residues. For each probe multiphasic kinetics were observed. The burial of tyrosine residues, as determined by the change in absorbance at 286 nm, revealed four phases, whereas the kinetics of refolding monitored by fluorescence revealed only two phases. The rates of the transients detected by fluorescence were independent of pH. One of the faster transients detected by delta A286 involved a decrease in absorbance, which is consistent with solvent exposure, rather than burial, and suggests the possibility of an abortive partially folded intermediate in the earlier stages of folding. Double-jump unfolding assays were used to follow the buildup and decay of an intermediate in the refolding reaction at -15 degrees C. At both pH* 3.0 and pH* 6.0 the maximum concentration of the intermediate was 25-30% of the total protein. The existence of a second pathway of slow folding was inferred from the difference in rate of formation of native enzyme and breakdown of the observed intermediate, and by computer simulations. In addition, the unfolding assay demonstrated that 20% of the unfolded protein was converted to native at a much faster rate, consistent with observations in aqueous solution that 80% of unfolded ribonuclease A consists of slow-folding species. Kinetics and amplitude data from these and other refolding experiments with different probes were used to develop possible models for the pathway of refolding. The simplest system consistent with the results for the slow-refolding species involves two parallel pathways with multiple intermediates on each of them. Several independent lines of evidence indicate that about 30% of the unfolded state refolds by the minor pathway, in which the slowest observed phase is attributed to the isomerization of Pro-93. The major pathway involves 50% of the unfolded state; the reason why it refolds slowly is not apparent. A native-like intermediate is formed considerably more rapidly in the major slow-refolding pathway, compared to the minor pathway.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Dissecting the non-specific and specific components of the initial folding reaction of barstar by multi-site FRET measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38-55 and 51-69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein (U) to unfolded protein in refolding conditions (U(C)). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate (I(E)). FRET efficiency and distance change differently for the different donor-acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U(C) and I(E) does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U(C) and to I(E) are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I(E), possesses specific structure.     

Kinetics and mechanism of the refolding of denatured ribonuclease A

On the basis of two experimental observations, it is established that the refolding mechanism of ribonuclease A (RNase A) is independent of the nature of the denaturant used [urea or guanidine hydrochloride (Gdn.HCl)]. First, by use of a double-jump technique, it is demonstrated that a similar nativelike intermediate exists on the major slow-folding pathway of both urea- and Gdn.HCl-denatured RNase A. Second, from the temperature dependence of the slow-refolding kinetics, it is shown that the activation parameters (both enthalpy and entropy) of the rate-limiting steps, as monitored by tyrosine absorbance and fluorescence, are identical for the refolding of urea- and Gdn.HCl- denatured RNase A. A refolding scheme involving one intermediate on each of the two slow-folding pathways is proposed by adopting the notion that RNase A refolds through a sequential mechanism. However, these two intermediates are formed from their respective unfolded forms (USII and USI) through two different processes of distinct physical origin. The intermediate IN, which is formed from the major slow-folding species USII through a conformational folding step, already possesses many properties of the native protein. In contrast, the intermediate (designated as I') on the minor slow-folding pathway is formed from USI by the isomerization of a proline residue (possibly Pro93) and is still conformationally unfolded. It is shown that such a refolding scheme can account for the known kinetic features of both major and minor slow-refolding pathways of RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin

The reversible unfolding and refolding kinetics of alpha-lactalbumin induced by concentration jump of guanidine hydrochloride were measured at pH 7.0 and 25 degrees C using tryptophan absorption at 292 nm, with varying concentrations of the denaturant and free Ca2+. The refolding reaction of alpha-lactalbumin from the fully unfolded (D) state occurs through the two stages: (1) instantaneous formation of a compact intermediate (the A state) that has a native-like secondary structure; (2) tight packing of the preformed secondary structure segments to lead finally to the native structure, this stage being the rate-determining step of the reaction and associated with acquisition of the specific structure necessary for strong Ca2+ binding. Under strongly native conditions, the observed kinetics of refolding is also complicated by the presence of a slow-folding species (10%) in the unfolded state. Considering these facts, the microscopic rate constants in folding and unfolding directions have been evaluated from the observed kinetics and from the equilibrium constants of the transitions among the native (N), A and D states. Close linear relationships have been found in the plots of the activation free energies, obtained from the microscopic rate constants, against the denaturant concentration. They are similar to the linear relationship between the free energy of unfolding and the denaturant concentration. It was demonstrated that the slope of the plots should be approximately proportional to a change in accessible surface area of the protein during the respective activation process, and that only a third of the difference in accessible surface area between A and N is buried in the critical activated state of folding. However, the selective effect of Ca2+ binding on the folding rate constant has been observed also, demonstrating that the specific Ca2+-binding substructure in the N state is already organized in the activated state. Thus, only a part of the protein molecule involving the Ca2+-binding region is organized in the activated state, with the other part of the molecule being left less organized, suggesting that the second stage of folding may be a sequential growing process of organized assemblage of the performed secondary structure segments.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.