Mutagenic analysis of the interior packing of an .alpha./.beta. barrel protein. Effects on the stabilities and rates of interconversion of the native and partially folded forms of the .alpha. subunit of tryptophan synthase

Abstract

A series of single and double amino acid replacements in four beta strands of the alpha subunit of tryptophan synthase from Salmonella typhimurium, and alpha/beta barrel protein, was made to study the interior packing of the barrel and to clarify its folding mechanism. The urea-induced unfolding of the alpha subunit is thought to involve a stable intermediate in which the amino folding unit (residues 1-188; helices 0-5, strands 1-6) remains folded while the carboxy folding unit (residues 189-268; helices 6-8, strands 7-8) becomes disordered [Beasty, A. M., & Matthews, C. R. (1985) Biochemistry 24, 3547; Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586]. Mutations in strands 1 (A18G and A18V), 6 (Y175Q), 7 (L209V), and 8 (G230A, G230V, and I232V) at the interface between these two folding units show that the effects on the stabilities of the native and intermediate conformations critically depend on the site of the replacement. Although all of these mutations decrease the stability of the native conformation, only the replacements in strand 6, Y175Q, and possibly strand 8, I232V, also perturb the intermediate. Comparisons of the effects of three pairs of double mutants with the effects of the constituent single mutants on stability show that strands 6 and 7 interact in both the intermediate and native conformations, while strands 1 and 8 interact only in the native conformation. Kinetic studies of unfolding indicate that the interactions which occur in the native conformation arise in the preceding transition state. These results demonstrate that the carboxy folding unit adopts an organized structure in the intermediate, contrary to our previous interpretation. The general implication is that the state of folding of one segment of a protein can depend on the presence of another, more stable element of structure.

On the Role of Helix 0 of the Tryptophan Synthetase α Chain of

“…TrpA proteins overexpressed in this strain are recoverable from insoluble inclusion bodies by denaturation in 6 M urea and renaturation by dialysis (31). Deletion TrpA proteins treated in this manner are soluble and stable.…”

“…6). Active TrpA species was separated from other forms of refolded or disordered TrpA proteins by chromatography on a DEAE-cellulose column (31). The first peak to elute generally had the highest TrpA-specific activity.…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…The complex HX properties of the peptides derived from the mixture of the I a and I b species at 5 M urea previously led to the conclusion that I a is structured in the β 2 (βα) 3-5 β6 region and I b has an expanded structure that includes α 1 , α 6 and β 7 . 24 The different protection observed for peptides [36][37][38][39][40][41][42][43][44][45][46][47] To test whether these differences reflect the different HX pulse regimes, the equilibrium intermediate at 5 M D 4 -urea, as a control, was also labeled with the same quenched-flow parameters as the burst-phase intermediate, i.e., a 50 ms pulse-label at pH 9.5 and 25 °C. The same protection pattern was obtained as with the 5 s pulse at pH 7.8 ( Figure 4).…”

“…Less populated states (B,C and E) may be more easily studied with experiments which probe transition states, such as ϕ-values. 45 Are nonproductive trajectories the result of topological frustration? Observing the nonproductive (yellow trace) trajectory in Figure 7A, this possibility seems likely since the trajectory appears to make short attempts forward to Q~425 but then falls back to Q~375.…”

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

“…Mutational methods have also been applied to the analysis of intermediates in the folding of several proteins, including the cu-subunit of tryptophan synthetase (Beasty et al, 1986;Tsuji et al, 1993), dihydrofolate reductase (Garvey et al, 1989;Perry et al, 1989), barnase (Matouschek et al, 1990;Serrano et al, 1992;Fersht 1995), and bovine pancreatic trypsin inhibitor (BPTI) Zhang & Goldenberg, 1993). These studies have shown that amino acid replacements can have clearly distinguishable effects on different steps in a folding mechanism, and have facilitated the identification of residues that contribute to the formation of particular intermediates.…”