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.     

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.     

C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer

The tailspike protein from the bacteriophage P22 is a well characterized model system for folding and assembly of multimeric proteins. Folding intermediates from both the in vivo and in vitro pathways have been identified, and both the initial folding steps and the protrimer-to-trimer transition have been well studied. In contrast, there has been little experimental evidence to describe the assembly of the protrimer. Previous results indicated that the C terminus plays a critical role in the overall stability of the P22 tailspike protein. Here, we present evidence that the C terminus is also the critical assembly point for trimer assembly. Three truncations of the full-length tailspike protein, TSPΔN, TSPΔC, and TSPΔNC, were generated and tested for their ability to form mixed trimer species. TSPΔN forms mixed trimers with full-length P22 tailspike, but TSPΔC and TSPΔNC are incapable of forming similar mixed trimer species. In addition, mutations in the hydrophobic core of the C terminus were unable to form trimer in vivo. Finally, the hydrophobic-binding dye ANS inhibits the formation of trimer by inhibiting progression through the folding pathway. Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer.     

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.     

Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike

The predominantly beta-sheet phage P22 tailspike adhesin contains eight reduced cysteines per 666 residue chain, which are buried and unreactive in the native trimer. In the pathway to the native trimer, both in vivo and in vitro transient interchain disulfide bonds are formed and reduced. This occurs in the protrimer, an intermediate in the formation of the interdigitated beta-sheets of the trimeric tailspike. Each of the eight cysteines was replaced with serine by site-specific mutagenesis of the cloned P22 tailspike gene and the mutant genes expressed in Escherichia coli. Although the yields of native-like Cys>Ser proteins varied, sufficient soluble trimeric forms of each of the eight mutants accumulated to permit purification. All eight single Cys>Ser mature proteins maintained the high thermostability of the wild type, as well as the wild-type biological activity in forming infectious virions. Thus, these cysteine thiols are not required for the stability or activity of the native state. When their in vivo folding and assembly kinetics were examined, six of the mutant substitutions--C267S, C287S, C458S, C613S, and C635S--were significantly impaired at higher temperatures. Four--C290S, C496, C613S, and C635--showed significantly impaired kinetics even at lower temperatures. The in vivo folding of the C613S/C635S double mutant was severely defective independent of temperature. Since the trimeric states of the single Cys>Ser substituted chains were as stable and active as wild type, the impairment of tailspike maturation presumably reflects problems in the in vivo folding or assembly pathways. The formation or reduction of the transient interchain disulfide bonds in the protrimer may be the locus of these kinetic functions.     

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.     

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.     

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.     

Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540-548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540-544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.     

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.     

Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation.

Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of m-values and delta Cp for these variants will be discussed in this context.     

Genetic and biochemical analysis of in vivo protein folding and subunit assembly

The in vivo pathway of folding and subunit assembly of a trimeric bacteriophage protein has been studied by characterizing precursors to the native protein and by analyzing temperature-sensitive mutations that kinetically block the pathway. The native trimer is formed via an intermediate composed of three partially folded chains, the protrimer. At 39°C, temperature-sensitive mutations prevent the formation of both the native trimer and the protrimer, possibly by destabilizing earlier intermediates. However, the mutations do not affect the stability of the native protein, formed at 30°C. Thus, these mutations identify amino acid residues involved in interactions that determine the folding pathway.     

Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants

Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type α-lactalbumin in future studies.     

Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants.

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core.

The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

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.     

Mutational analysis of the BPTI folding pathway: II. Effects of aromatic-->leucine substitutions on folding kinetics and thermodynamics.

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.     

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.