Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike

Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer. The effects of pressure on two different tsf mutants, G244R and E196K, were explored. Pressure treatment of both G244R and E196K aggregates produced a folded trimer. E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein. In contrast, trimer formation from pressure-treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment. The center-of-mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates. Taken together, these results suggest that E196K has a primary defect in formation of the beta-helix during monomer collapse, while G244R is primarily an assembly defect.     

Maximizing recovery of native protein from aggregates by optimizing pressure treatment

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.     

The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.     

Secondary structure and thermostability of the phage P22 tailspike. XX. Analysis by Raman spectroscopy of the wild-type protein and a temperature-sensitive folding mutant

The thermostable tailspike endorhamnosidase of bacteriophage P22 has been investigated by laser Raman spectroscopy to determine the protein's secondary structure and the basis of its thermostability. The conformation of the native tailspike, determined by Raman amide I and amide III band analyses, is 52 to 61% beta-sheet, 24 to 27% alpha-helix, 15 to 21% beta-turn and 0 to 10% other structure types. The secondary structure of the wild-type tailspike, as monitored by the conformation-sensitive Raman amide bands, was stable to 80 degrees C, denatured reversibly between 80 and 90 degrees C, and irreversibly above 90 degrees C. The purified native form of a temperature-sensitive folding mutant (tsU38) contains secondary structures virtually identical to those in the wild-type in aqueous solution at physiological conditions (0.05 M-Na+ (pH 7.5], at both permissive (20 degrees C) and restrictive (40 degrees C) temperatures. This supports previous results showing that the mutational defect at 40 degrees C affects intermediates in the folding pathway rather than the native structure. At temperatures above 60 degrees C the wild-type and mutant forms were distinguishable: the reversible and irreversible denaturation thresholds were approximately 15 to 20 degrees C lower in the mutant than in the wild-type protein. The irreversible denaturation of the mutant tailspikes led to different aggregation/polymerization products from the wild-type, indicating that the mutation altered the unfolding pathway. In both cases only a small percentage of the native secondary structure was altered by irreversible thermal denaturation, indicating that the aggregated states retain considerable native structure.     

Hydrostatic pressure rescues native protein from aggregates.

Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods.     

P22 tailspike trimer assembly is governed by interchain redox associations

Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.     

Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein

P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium. The 70 kDa subunits include long beta-helix domains. After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts. Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms. The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure. We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins. The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding. Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer. By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge. Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains. These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains. This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Conformational stability of P22 tailspike proteins carrying temperature-sensitive folding mutations

The thermostable tailspike endorhamnosidase of Salmonella phage P22 provides a model system for comparing the role of amino acid sequences in determining the intracellular folding pathway with their role in stabilizing the mature structural protein. Complete Raman band assignments are given here for the native form of the tailspike trimer in aqueous solution. Once correctly folded and assembled, the wild-type and two well-characterized mutant proteins, tsfIle258----Leu and tsfGly323----Asp, exhibit the same secondary structure in solution, consisting predominantly of beta-strand (56 +/- 5%) and turns (17 +/- 2%). Raman bands that are sensitive indicators of hydrogen-bonding interactions of tyrosine (phenolic OH) and tryptophan (indole NH) are unchanged between 30 and 80 degrees C in both wild type and tsf mutants. Similarly, Raman bands that are sensitive to changes in the hydrophobic environment of nonpolar side chains exhibit no significant temperature dependence in wild type and tsf mutants. In contrast, these conformational features are greatly altered by chemical denaturation of the tailspike with lithium halide and guanidine hydrochloride. In the chemically denatured tailspike, the beta-strand structure is substantially converted to irregular or "random coil" conformation. These findings confirm conclusions from physiological studies that the three-dimensional structures of the tsf mutants, once stabilized at permissive temperatures, are equivalent to the native structure of the wild type, and this structure is maintained at temperatures far above those that block the folding of the chain into the final native conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An aggregation-prone intermediate species is present in the unfolding pathway of the monomeric portal protein of bacteriophage P22: implications for portal assembly

The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.     

Mechanism of phage P22 tailspike protein folding mutations.

Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.     

Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.     

Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.     

Formation of fibrous aggregates from a non-native intermediate: the isolated P22 tailspike beta-helix domain.

In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel beta-helix, is essentially structured in this species. We used the isolated beta-helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.     

Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. A model for inclusion body formation

The in vivo accumulation of polypeptide chains in the form of aggregated non-native states is a problem in many applications of biotechnology. In the maturation pathway of the thermostable P22 tailspike endorhamnosidase, the folding and chain association intermediates can be distinguished from the native tailspikes in crude extracts of phage-infected Salmonella cells. Temperature-sensitive folding mutations, at many sites in the chain, destabilize these conformational intermediates preventing the formation of native tailspikes at restrictive temperatures (Goldenberg, D. P., Smith, D. H., and King, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7060-7064). We report here that both wild type and mutant tailspike polypeptide chains which fail to reach the native state accumulate in an aggregated state. These off-pathway aggregates form from a thermolabile intermediate in the productive folding pathway. These aggregation reactions are suppressed by lowering the temperature of maturation. Similar off-pathway steps from folding intermediates may account for the non-native aggregates often found in the expression of cloned genes in heterologous hosts.     

Pressure effect on the temperature-induced unfolding and tendency to aggregate of myoglobin

This work demonstrates that pressure-induced partially unfolded states play a very important role in the aggregation of proteins. The high-pressure unfolding of horse heart metmyoglobin results in an intermediate form that shows a strong tendency to aggregate after pressure release. These aggregates are similar to those that are usually observed upon temperature denaturation. Infrared spectra in the amide I region indicate the formation of an intermolecular antiparallel beta-sheet stabilized by hydrogen bonding. The formation of the aggregates is temperature-dependent. Below 30 degrees C, no aggregation is taking place as seen from the infrared spectra. At 45 and 60 degrees C, two types of aggregates are formed: one that can be dissociated by moderate pressures and one that is pressure-insensitive. When precompressed at 5 degrees C, temperature-induced aggregation takes place at lower temperature (38 degrees C) than without pressure pretreatment (74 degrees C).     

A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase.

With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.