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The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

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.

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.

Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been viewed as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsid assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure coat protein monomers are mixed with scaffolding protein. We show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding protein stripped out) were diluted so that the concentration was below the dissociation constant ( approximately 5 microM) for coat protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the released coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented illustrate that coat protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and that the reaction follows the law of mass action.

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.

Icosahedral capsid assembly is an example of a reaction controlled solely by the interactions of the proteins involved. Bacteriophage P22 procapsids can be assembled in vitro by mixing coat and scaffolding proteins in a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here, we investigated the effect of the buffer composition on the interactions necessary for capsid assembly. Different concentrations of various salts, chosen to follow the electroselectivity series for anions, were added to the assembly reaction. The concentration and type of salt was found to be crucial for proper nucleation of procapsids. Nucleation in low salt concentrations readily occurred but led to bowl-like partial procapsids, as visualized by negative stain electron microscopy. The edge of the partial capsids remained assembly-competent since coat protein addition triggered procapsid completion. The addition of salt to the partial capsids also caused procapsid completion. In addition, each salt affected both assembly rates and the extent of procapsid formation. We hypothesize that low salt conditions increase the coat protein:scaffolding protein affinity, causing excessive nuclei to form, which decreases coat protein levels leading to incomplete assembly.

Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second-site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. Here we report evidence that su substitutions that rescue bacteriophage P22 temperature-sensitive-folding (tsf) coat protein variants function in a novel way. The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production. The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents. Phage-infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones. Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents. We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone-driven kinetic partitioning between aggregation-prone intermediates and the final assembled state. Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect.

SecA, a 202 kDa dimeric protein, is the ATPase for the Sec-dependent translocase of precursor proteins in vivo. SecA must undergo conformational changes, which may involve dissociation into a monomer, as it translocates the precursor protein across the inner membrane. To better understand the dynamics of SecA in vivo, protein folding studies to probe the native, intermediate, and unfolded species of SecA in vitro have been done. SecA folds through a stable dimeric intermediate and dimerizes in the dead-time of a manual-mixing kinetic experiment ( approximately 5-7 seconds). Here, stopped-flow fluorescence and CD, as well as ultra-rapid continuous flow fluorescence techniques, were used to further probe the rapid folding kinetics of SecA. In the absence of urea, rapid, near diffusion-limited ( approximately 10(9)M(-1)s(-1)) SecA dimerization occurs following a rate-limiting unimolecular rearrangement of a rapidly formed intermediate. Multiple kinetic folding and unfolding phases were observed and SecA was shown to have multiple native and unfolded states. Using sequential-mixing stopped-flow experiments, SecA was determined to fold via parallel channels with sequential intermediates. These results confirm that SecA is a highly dynamic protein, consistent with the rapid, major conformational changes it must undergo in vivo.

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.

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.

To assemble into a virus with icosahedral symmetry, capsid proteins must be able to attain multiple conformations. Whether this conformational diversity is achieved during folding of the subunit, or subsequently during assembly, is not clear. Phage P22 coat protein offers an ideal model to investigate the folding of a monomeric capsid subunit since its folding is independent of assembly. Our early studies indicated that P22 coat protein monomers could be folded into an assembly-competent state in vitro, with evidence of a kinetic intermediate. Using urea denaturation, coat protein monomers are shown to be marginally stable. The reversible folding of coat protein follows a three-state model, N if I if U, with an intermediate exhibiting most of the tryptophan fluorescence of the folded state, but little secondary structure. Folding and unfolding kinetics monitored by circular dichroism, tryptophan fluorescence, and bisANS fluorescence indicate that several kinetic intermediates are populated sequentially through parallel channels en route to the native state. Additionally, two native states were identified, suggesting that the several conformers required to assemble an icosahedral capsid may be found in solution before assembly ensues.

Bacteriophage assembly frequently begins with the formation of a precursor capsid that serves as a DNA packaging machine. The DNA packaging is accompanied by a morphogenesis of the small round precursor capsid into a large polyhedral DNA-containing mature phage. In vitro, this transformation can be induced by heat or chemical treatment of P22 procapsids. In this work, we examine bacteriophage P22 morphogenesis by comparing three-dimensional structures of capsids expanded both in vitro by heat treatment and in vivo by DNA packaging. The heat-expanded capsid reveals a structure that is virtually the same as the in vivo expanded capsid except that the pentons, normally present at the icosahedral fivefold positions, have been released. The similarities of these two capsid structures suggest that the mechanism of heat expansion is similar to in vivo expansion. The loss of the pentons further suggests the necessity of specific penton-hexon interactions during expansion. We propose a model whereby the penton-hexon interactions are stabilized through interactions of DNA, coat protein, and other minor proteins. When considered in the context of other studies using chemical or heat treatment of capsids, our study indicates that penton release may be a common trend among double-stranded DNA containing viruses.

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.

Though many proteins in the cell are large and multimeric, their folding has not been extensively studied. We have chosen SecA as a folding model because it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent preprotein translocase of many bacteria. SecA is a soluble protein that can penetrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functions in vivo. Kinetic folding transitions in the presence of urea were monitored using circular dichroism and tryptophan fluorescence, while equilibrium folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition exhibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N(2) if I(2) if 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. The SecA dimer was determined to have an overall stability (DeltaG) of -22.5 kcal/mol. We also investigated the stability of SecA using analytical ultracentrifugation equilibrium and velocity sedimentation, which again indicated that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the native dimer. Unfolding of native, dimeric SecA was slow with a relaxation time in H(2)O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.

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.

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.

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.

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.

Our present understanding of the action of the chaperonins GroEL/S on protein folding is based primarily on in vitro studies, whereas the folding of proteins in the cellular milieu has not been as thoroughly investigated. We have developed a means of examining in vivo protein folding and assembly that utilizes the coat protein of bacteriophage P22, a naturally occurring substrate of GroEL/S. Here we show that amino acid substitutions in coat protein that cause a temperature-sensitive-folding (tsf) phenotype slowed assembly rates upon increasing the temperature of cell growth. Raising cellular concentrations of GroEL/S increased the rate of assembly of the tsf mutant coat proteins to nearly that of wild-type (WT) coat protein by protecting a thermolabile folding intermediate from aggregation, thereby increasing the concentration of assembly-competent coat protein. The rate of release of the tsf coat proteins from the GroEL/S-coat protein ternary complex was approximately 2-fold slower at non-permissive temperatures when compared with the release of WT coat protein. However, the rate of release of WT or tsf coat proteins at each temperature remained constant regardless of GroEL/S levels. Thus, raising the cellular concentration of GroEL/S increased the amount of assembly-competent tsf coat proteins not by altering the rates of folding but by increasing the probability of GroEL/S-coat protein complex formation.

Three cold-sensitive mutants in phage P22 coat protein have been characterized to determine the effects of the amino acid substitutions that cause cold sensitivity on the folding pathway and the conformation of refolded coat protein. Here we find that the three cold-sensitive mutants which have the threonine residue at position 10 changed to isoleucine (T10I), the arginine residue at position 101 changed to cysteine (R101C), or the asparagine residue at position 414 changed to serine (N414S) were capable of folding from a denatured state into a soluble monomeric species, but in each case, the folded conformation was altered. Changes in the kinetics of folding were observed by both tryptophan and bisANS fluorescence. In contrast to the temperature-sensitive for folding coat protein mutants which can be rescued at nonpermissive temperatures in vivo by the overproduction of molecular chaperones GroEL and GroES [Gordon, C. L., Sather, S. K., Casjens, S., & King, J. (1994) J. Biol. Chem. 269, 27941-27951], the folding defects associated with the cold-sensitive amino acid substitutions were not recognized by GroEL and GroES.

Cold-sensitive mutations in phage P22 coat protein cause the accumulation of precursor capsids in cells growing at the nonpermissive temperature (16 degrees C). The assembly of coat proteins which carry the substitutions threonine at position 10 to isoluecine (T10I), arginine at position 101 to cysteine (R101C), or asparagine at position 414 to serine (N414S) which cause cold-sensitivity has been investigated. All three proteins were found to fold into a monomeric species. Coat proteins carrying the amino acid substitutions T10I and R101C were not able to interact with scaffolding protein appropriately to initiate assembly in vitro while coat protein carrying the substitution N414S was able to assemble; however, capsids formed of this protein had an increased affinity for scaffolding protein. These amino acid substitutions define two regions in coat protein that are essential for the interaction of coat protein with scaffolding protein at different stages in capsid maturation.

The coat protein that forms the icosahedral shell of phage P22 can be efficiently refolded in vitro [Teschke, C. M., & King, J. (1993) Biochemistry 32, 10839-10847]. Temperature-sensitive mutants of coat protein interfere with folding or assembly in vivo [Gordon, C. L., & King, J. (1993) J. Biol. Chem. 268, 9358-9368]. The folding of a set of phage P22 coat proteins carrying the temperature-sensitive for folding (tsf) substitutions W48Q, A108V, G232D, T294I, and F353L has been investigated in vitro. Denatured tsf polypeptides were able to fold into soluble species with high efficiency. The efficiency of folding of the wild-type (WT) and mutant polypeptides at different temperatures showed sharp transitions where aggregation predominated over folding. The refolding of the tsf mutant proteins did not show an obvious thermal defect in yield. The tsf polypeptides folded through the long-lived kinetic intermediate previously described for WT coat protein with similar relaxation times. The folding kinetics of the tsf polypeptides in bisANS, a hydrophobic fluorescent dye, were also similar to those of the WT protein. However, the folded tsf proteins showed decreased secondary structure compared to WT coat protein. Analysis of the folded state by native gel electrophoresis revealed that the tsf coat proteins folded into dimers and trimers, not monomers. The dimer and trimer species were incompetent for assembly. Once formed, dimers and trimers showed no propensity toward aggregation. The folding pathway of the mutant polypeptides must be quite similar to the WT pathway, but at some step inappropriate intersubunit interactions occur due to the amino acid substitutions, trapping the subunits from assembly.

Bacteriophage P22 is a double-stranded DNA containing phage. Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid. The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously [Prevelige, P. E., King, J., & Silva, J. L. (1994) Biophys. J. 66, 1631-1641]. The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar. However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods. While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 degrees C), the G232D mutant shells showed time-dependent dissociation under these conditions. Decreasing the temperature to 1 degree C dramatically accelerated the dissociation of G232D mutant under applied pressure. On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Within infected Salmonella cells, newly synthesized 47-kDa phage P22 coat polypeptides fold without covalent modifications into assembly-competent subunits. Coat protein subunits interact with scaffolding protein to form the icosahedral procapsid precursor of the mature, T = 7, virions. In these lattices, the coat subunits form seven classes of local bonding interactions [Prasad, B. V. V., Prevelige, P. E., Marieta, E., Chen, R. O., Thomas, D., King, J., & Chiu, W. (1993) J. Mol. Biol. 231, 65-74]. Coat protein denatured in guanidine hydrochloride could be refolded to soluble, monomeric subunits by rapid dilution into buffer at concentrations of protein up to 25 micrograms/mL. The fluorescence emission spectrum of soluble coat protein monomers was between that of the assembled shells and the denatured protein, suggesting the presence of tryptophans at the subunit interfaces in the shells. Kinetic studies of the refolding of coat protein revealed an intermediate whose continued folding could be inhibited by the hydrophobic dye bisANS. The kinetic intermediate bound 10.80 +/- 1.20 bisANS molecules while the folded monomer bound 1.24 +/- 0.36 bisANS molecules. When coat polypeptide chains were refolded at 50 micrograms/mL, aggregation competed with folding. Aggregation of the folding intermediates increased in the presence of bisANS. The kinetic folding intermediate that binds bisANS probably represents the species at the junction of the productive pathway to soluble and assembly-competent coat monomers and the off-pathway steps to inclusion bodies. The relationship between these soluble monomers and the conformations observed in the T = 7 lattice remains unclear.

The precursor shells of dsDNA bacteriophages are assembled by the polymerization of competent states of coat and scaffolding subunits. The fluorescent dye 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid) (bisANS) binds to both the coat and scaffolding proteins from the Salmonella typhimurium bacteriophage P22. It displays little affinity for the polymerized forms of the proteins. The subunits with bound bisANS are incapable of assembling into procapsids. The binding constants of bisANS for both coat and scaffolding protein monomers have been measured and are 7 and 6 microM, respectively. Binding of bisANS to coat protein has little effect on the conformation as determined by circular dichroism and susceptibility to proteolysis. Binding of bisANS to scaffolding protein induces a change in the secondary structure consistent with a loss of alpha-helix, and an altered susceptibility to proteolysis. We suggest that the bisANS is probably binding at sites responsible for intersubunit interactions and thereby inhibiting capsid assembly.

High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies. This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation. Methods for optimizing the yield of correctly assembled oligomers are discussed.

It has been proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export. As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export. (Liu, G., Topping, T. B., Cover, W. H., and Randall, L. L. (1988) J. Biol. Chem. 263, 14790-14793). Here we describe mutations of the gene encoding ribose-binding protein that were selected as suppressors of a defect in export of that protein and that alter the folding pathway. We propose that selection of such suppressors may provide a general method to obtain mutations that affect the folding properties of any protein that can be expressed and exported in E. coli.