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 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.

SecA folds via a dimeric intermediate.

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.

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.

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.

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.

GroEL and GroES control of substrate flux in the in vivo folding pathway of phage P22 coat protein.

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.

The folded conformation of phage P22 coat protein is affected by amino acid substitutions that lead to a cold-sensitive phenotype.

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.

Interactions between coat and scaffolding proteins of phage P22 are altered in vitro by amino acid substitutions in coat protein that cause a cold-sensitive phenotype.

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.

In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity.

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.

Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability.

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)

Inhibition of viral capsid assembly by 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid).

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.

Folding of the phage P22 coat protein in vitro.

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.

Folding and assembly of oligomeric proteins in Escherichia coli.

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.

Mutations that affect the folding of ribose-binding protein selected as suppressors of a defect in export in Escherichia coli.

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.

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes.

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.