A concerted mechanism for the suppression of a folding defect through interactions with chaperones

Specific amino acid substitutions confer a temperature-sensitive-folding (tsf) phenotype to bacteriophage P22 coat protein. Additional amino acid substitutions, called suppressor substitutions (su), relieve the tsf phenotype. These su substitutions are proposed to increase the efficiency of procapsid assembly, favoring correct folding over improper aggregation. Our recent studies indicate that the molecular chaperones GroEL/ES are more effectively recruited in vivo for the folding of tsf:su coat proteins than their tsf parents. Here, the tsf:su coat proteins are studied with in vitro equilibrium and kinetic techniques to establish a molecular basis for suppression. The tsf:su coat proteins were monomeric, as determined by velocity sedimentation analytical ultracentrifugation. The stability of the tsf:su coat proteins was ascertained by equilibrium urea titrations, which were best described by a three-state folding model, N <--> I <--> U. The tsf:su coat proteins either had stabilized native or intermediate states as compared with their tsf coat protein parents. The kinetics of the I <--> U transition showed a decrease in the rate of unfolding and a small increase in the rate of refolding, thereby increasing the population of the intermediate state. The increased intermediate population may be the reason the tsf:su coat proteins are aggregation-prone and likely enhances GroEL-ES interactions. The N --> I unfolding rate was slower for the tsf:su proteins than their tsf coat parents, resulting in an increase in the native state population, which may allow more competent interactions with scaffolding protein, an assembly chaperone. Thus, the suppressor substitution likely improves folding in vivo through increased efficiency of coat protein-chaperone interactions.     

Alleviation of a defect in protein folding by increasing the rate of subunit assembly

Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.     

GroEL/S substrate specificity based on substrate unfolding propensity

Phage P22 wild-type (WT) coat protein does not require GroEL/S to fold but temperature-sensitive-folding (tsf) coat proteins need the chaperone complex for correct folding. WT coat protein and all variants absolutely require P22 scaffolding protein, an assembly chaperone, to assemble into precursor structures termed procapsids. Previously, we showed that a global suppressor (su) substitution, T1661, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf:T1661 coat protein:GroEL complexes compared with the tsf parents. The increased concentration of complexes resulted in more assembly-competent coat proteins because of a shift in the chaperone-driven kinetic partitioning between aggregation-prone intermediates toward correct folding and assembly. We have now investigated the folding and assembly of coat protein variants that carry a different global su substitution, F170L. By monitoring levels of phage production in the presence of a dysfunctional GroEL we found that tsf:F170L proteins demonstrate a less stringent requirement for GroEL. Tsf:F170L proteins also did not cause induction of the chaperones. Circular dichroism and tryptophan fluorescence indicate that the native state of the tsf: F170L coat proteins is restored to WT-like values. In addition, native acrylamide gel electrophoresis shows a stabilized native state for tsf:F170L coat proteins. The F170L su substitution also increases procapsid production compared with their tsf parents. We propose that the F170L su substitution has a decreased requirement for the chaperones GroEL and GroES as a result of restoring the tsf coat proteins to a WT-like state. Our data also suggest that GroEL/S can be induced by increasing the population of unfolding intermediates.     

Single amino acid substitutions globally suppress the folding defects of temperature-sensitive folding mutants of phage P22 coat protein.

The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 coat protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of coat protein. Additional amino acid residues that are critical to the folding process of P22 coat protein were identified by isolating second site suppressors of the tsf coat proteins. Suppressor substitutions isolated from the phage carrying the tsf coat protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf coat protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the coat protein sequence and were 8-190 amino acid residues away from the tsf parent. Although the folding of coat proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of coat protein.     

Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching

Eighteen single amino acid substitutions in phage P22 coat protein cause temperature-sensitive folding defects (tsf). Three intragenic global suppressor (su) substitutions (D163G, T166I and F170L), localized to a flexible loop, rescue the folding of several tsf coat proteins. Here we investigate the su substitutions in the absence of the original tsf substitutions. None of the su variant coat proteins displayed protein folding defects. Individual su substitutions had little effect on phage production in vivo; yet double and triple combinations resulted in a cold-sensitive (cs) phenotype, consistent with a defect in assembly. During virus assembly and maturation, conformational switching of capsid subunits is required when chemically identical capsid subunits form an icosahedron. Analysis of double- and triple-su phage-infected cell lysates by negative-stain electron microscopy reveals an increase in aberrant structures at the cs temperature. In vitro assembly of F170L coat protein causes production of polyheads, never seen before in phage P22. Purified procapsids composed of all of the su coat proteins showed defects in expansion, which mimics maturation in vitro. Our results suggest that a previously identified surface-exposed loop in coat protein is critical in conformational switching of subunits during both procapsid assembly and maturation.     

GroEL binds a late folding intermediate of phage P22 coat protein

GroEL recognizes proteins that are folding improperly or that have aggregation-prone intermediates. Here we have used as substrates for GroEL, wildtype (WT) coat protein of phage P22 and 3 coat proteins that carry single amino acid substitutions leading to a temperature-sensitive folding (tsf) phenotype. In vivo, WT coat protein does not require GroEL for proper folding, whereas GroEL is necessary for the folding of the tsf coat proteins; thus, the single amino acid substitutions cause coat protein to become a substrate for GroEL. The conformation of WT and tsf coat proteins when in a binary complex with GroEL was investigated using tryptophan fluorescence, quenching of fluorescence, and accessibility of the coat proteins to proteolysis. WT coat protein and the tsf coat protein mutants were each found to be in a different conformation when bound to GroEL. As an additional measure of the changes in the bound conformation, the affinity of binding of WT and tsf coat proteins to GroEL was determined using a fluorescence binding assay. The tsf coat proteins were bound more tightly by GroEL than WT coat protein. Therefore, even though the proteins are identical except for a single amino acid substitution, GroEL did not bind these substrate polypeptides in the same conformation within its central cavity. Therefore, GroEL is likely to bind coat protein in a conformation consistent with a late folding intermediate, with substantial secondary and tertiary structure formed.     

Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.     

Aggregation and assembly of phage P22 temperature-sensitive coat protein mutants in vitro mimic the in vivo phenotype

Aggregation is a common side reaction in the folding of proteins which is likely due to inappropriate interactions of folding intermediates. In the in vivo folding of phage P22 coat protein, amino acid substitutions that cause a temperature-sensitive-folding (tsf) phenotype lead to the localization of the mutant coat proteins to inclusion bodies. Investigated here is the aggregation of wild-type (WT) coat protein and 3 tsf mutants of coat protein. The tsf coat proteins aggregated when refolded in vitro at high temperature. If the tsf coat proteins were refolded at 4 degrees C, they were able attain an assembly active state. WT coat protein, on the other hand, did not aggregate significantly even when folded at high temperature. The refolded tsf mutants exhibited altered secondary and tertiary structures and had an increased surface hydrophobicity, which may explain the increased propensity of their folding intermediates to aggregate.     

Surface amino acids as sites of temperature-sensitive folding mutations in the P22 tailspike protein

Temperature-sensitive folding (tsf) mutations in the gene for the thermostable P22 tailspike interfere with the polypeptide chain folding and association pathway at restrictive temperature without altering the thermostability of the protein once correctly folded and assembled at permissive temperature. Though the native proteins matured at permissive temperature are biologically active, many of them display alterations in electrophoretic mobility. The native forms of 15 of these tsf mutant proteins have been purified and characterized. The purified proteins differed in electrophoretic mobility and isoelectric point from wild type but did not show evidence of major conformational alterations. The results suggest that the electrophoretic variations conferred by the 15 tsf amino acid substitutions are due to changes in the net charge at solvent-accessible sites in the native form of the mutant protein. During the maturation of the chains at restrictive temperature, these sites influence the conformation of intermediates in chain folding and association. The amino acid sequences at these sites resemble those found at turns in polypeptide chains. The isolation of tsf mutations requires that the mature structure of the tailspike accommodates the mutant amino acid substitution without loss of function. The solvent-accessible sites are probably at the surface of this structural protein. This would explain how bulky mutant substitutions, such as arginines for glycines, are accommodated in the native tailspike structure. Such sites, stabilizing intermediates in the folding pathway and located on the surface of the mature protein, probably represent a general class of conformational substrates for tsf mutations.     

Amino acid substitutions influencing intracellular protein folding pathways.

Though an increasing variety of chaperonins are emerging as important factors in directing polypeptide chain folding off the ribosome, the primary amino acid sequence remains the major determinant of final conformation. The ability to identify cytoplasmic folding intermediates in the formation of the tailspike endorhamnosidase of phage P22 has made it possible to isolate two classes of mutations influencing folding intermediates-temperature-sensitive folding mutations and global suppressors of tsf mutants. These and related amino acid substitutions in eukaryotic proteins are discussed in the context of inclusion body formation and problems in the recovery of correctly folded proteins.     

Molecular properties of global suppressors of temperature-sensitive folding mutations in P22 tailspike endorhamnosidase.

Two global suppressors (Val-331 greater than Ala and Ala-334 greater than Val) have been identified for temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 (Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J., and King, J. (1991) Science 253, 54-58). We have introduced 19 different single amino acid substitutions at the two global suppressor sites independently and examined the effects on the tailspike formation in Escherichia coli. Folding and maturation patterns of the various substitutions at the two global suppressor sites in the wild-type background suggest that Val-331 is located on the protein surface and Ala-334 is in the hydrophobic region. In combination with a tsf mutation, tsfH304 (Gly-244 greater than Arg), only Gly at 331 and Ile at 334, the substitutions that have similar side chain properties to the original suppressor sequences, were active as tsf suppressors. The newly identified suppressors of tsfH304 could also alleviate the tsf defect of three other mutations. The mutant carrying both Val-331 greater than Ala and Ala-334 greater than Val substitutions was also a global suppressor and was more active in suppressing the tsf defect than mutants carrying only one substitution. The suppressors may act by increasing the stability of an intermediate in the productive pathway of folding and maturation of the mutant polypeptides.     

Folding defects caused by single amino acid substitutions in a subunit are not alleviated by assembly

Significant stabilization of a protein often occurs when it is assembled into an oligomer. Bacteriophage P22 contains 420 monomers of coat protein that are stabilized by the assembly and maturation processes. The effects of eight single amino acid substitutions in coat protein that each cause a temperature-sensitive-folding defect were investigated to determine if the conformational differences previously observed in the monomers could be alleviated by assembly or maturation. Several techniques including differential scanning calorimetry, heat-induced expansion, urea denaturation, and sensitivity to protease digestion were used to explore the effects of the amino acid substitutions on the conformation of coat protein, once assembled. Each of the amino acid substitutions caused a change in the conformation as compared to wild-type coat protein, observed by at least one of the probes used. Thus, neither assembly nor expansion entirely corrected the conformational defects in the monomeric subunits of the folding mutants.     

Nature and distribution of sites of temperature-sensitive folding mutations in the gene for the P22 tailspike polypeptide chain

Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.     

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.     

Identification of sites influencing the folding and subunit assembly of the P22 tailspike polypeptide chain using nonsense mutations

Amber mutations have been isolated and mapped to more than 60 sites in gene 9 of P22 encoding the thermostable phage tailspike protein. Gene 9 is the locus of over 30 sites of temperature sensitive folding (tsf) mutations, which affect intermediates in the chain folding and subunit association pathway. The phenotypes of the amber missense proteins produced on tRNA suppressor hosts inserting serine, glutamine, tryosine and leucine have been determined at different temperatures. Thirty-three of the sites are tolerant, producing functional proteins with any of the four amino acids inserted at the sites, independent of temperature. Tolerant sites are concentrated at the N-terminal end of the protein indicating that this region is not critical for conformation or function. Sixteen of the sites yield temperature sensitive missense proteins on at least one nonsense suppressing host. Most of the sites with ts phenotypes map to the central region of the gene which is also the region where most of the tsf mutations map. Mutations at 15 of the sites have a lethal phenotype on at least one tRNA suppressor host. For nine out of ten sites tested with at least one lethal phenotype, the primary defect was in the folding or subunit association of the missense polypeptide chain. This analysis of the tailspike missense proteins distinguishes three classes of amino acid sites in the polypeptide chain; residues whose side chains contribute little to folding, subunit assembly or function; residues critical for maintaining the folding and subunit assembly pathway at the high end of the temperature range of phage growth; and residues critical over the entire temperature range of growth.     

Isolation of suppressors of temperature-sensitive folding mutations.

Mutations in the tailspike gene (gene 9) of Salmonella typhimurium phage P22 have been used to identify amino acid interactions during the folding of a polypeptide chain. Since temperature-sensitive folding (tsf) mutations cause folding defects in the P22 tailspike polypeptide chain, it is likely that mutants derived from these and correcting the original tsf defects (second-site intragenic suppressors) identify interactions during the folding pathway. We report the isolation and identification of second-site revertants to tsf mutants.     

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.     

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.     

Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101.     

Limits of protein folding inside GroE complexes

The GroE chaperones of Escherichia coli promote the folding of other proteins under conditions where no spontaneous folding occurs. One requirement for this reaction is the trapping of the nonnative protein inside the chaperone complex. Encapsulation may be important to prevent unfavorable intermolecular interactions during folding. We show here that, especially for oligomeric proteins, the timing of encapsulation and release is of critical importance. If this cycle is decelerated, misfolding is observed inside functional chaperone complexes.