Mutations that Stabilize Folding Intermediates of Phage P22 Tailspike Protein: Foldingin Vivoandin Vitro, Stability, and Structural Context

Beissinger, M.; Lee, Sang Chul; Steinbacher, Stefan; Reinemer, Peter; Huber, Robert; Yu, Myeong-Hee; Seckler, Robert

doi:10.1006/jmbi.1995.0288

Cited by 37 publications

(28 citation statements)

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“…The tsf mutations also affect refolding of puri®ed polypeptide chains in vitro by destabilizing folding intermediates (Beiûinger et al, 1995;Mitraki et al, 1993). These results establish that the tsf mutations act directly in the chain folding pathway, and not through interaction with cellular components.…”

Section: Introductionmentioning

confidence: 98%

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Haase-Pettingell

King

1997

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 98%

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Haase-Pettingell

King

1997

Journal of Molecular Biology

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“…36 An analysis of the refolding pathways of wild-type tailspike and of a number of single-and doublemutant proteins at varied temperature has confirmed that both types of mutations act by altering the global stability of well-structured, native-like folding intermediates. 25,37,38 Accordingly, there is a good correlation between the effect of a mutation on tailspike folding and its effect on the stability of the finally folded structure.…”

Section: P22 Tailspike Proteinmentioning

confidence: 89%

“…36 This was explained by improved hydrophobic stacking mediated by the enlarged hydrophobic side chain in loosely structured intermediates on the one hand, but steric clashes in the tightly packed native structure on the other hand. 37 In addressing the question of whether the mutants investigated here might similarly act as suppressors of the tsf phenotype, we additionally created a double mutant containing both E359G and the well-characterized tsf mutation G244R. E359G, in combination with the tsf mutation, is not able to avoid the excessive aggregation of the destabilized intermediates at elevated temperature, and can thus not be classified as a suppressor mutation.…”

Section: Rational Mutations In the P22 Tailspike Proteinmentioning

confidence: 99%

“…This can be explained by the mutations having different or even reciprocal effects on the stability of early and late folding intermediates or the native structure. 37 Therefore, we tested this possibility for E359G, one of the mutants with presumptive release of backbone strain isolated here, as a representative example. The well-characterized tsf mutant G244R was chosen as a background, and the results of the refolding yields of wt, G244R and the double mutant G244R/E359G are illustrated in Figure 6.…”

Section: (H I); Where I(h I) Is the Intensity Of The Ith Measuremenmentioning

confidence: 99%

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Plasticity and steric strain in a parallel ?-helix: Rational mutations in the P22 tailspike protein

et al. 2000

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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.

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“…Relatively small changes such as single amino acid substitutions can disrupt the folding process and misfolded proteins that aggregate can result (Thomas et al 1995). To alleviate these folding defects, second site amino acid substitutions, termed suppressor substitutions (su), are able to revert the misfolding caused by the original substitution (Mitraki et al 1991;Beißinger et al 1995;Sideraki et al 2001). Sites of both the single amino acid substitutions and second site suppressor substitutions pinpoint areas in the protein that are crucial for the proper folding and stability of the molecule.…”

Section: Introductionmentioning

confidence: 99%

GroEL/S substrate specificity based on substrate unfolding propensity

Parent

Teschke

2007

Cell Stress Chaper

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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, T166I, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf :T166I 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.

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Mutations that Stabilize Folding Intermediates of Phage P22 Tailspike Protein: Foldingin Vivoandin Vitro, Stability, and Structural Context

Cited by 37 publications

References 0 publications

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Plasticity and steric strain in a parallel ?-helix: Rational mutations in the P22 tailspike protein

GroEL/S substrate specificity based on substrate unfolding propensity

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