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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.
Biochemistry. 1993 Jun 01; 32(21):5566-75.B

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.

Authors+Show Affiliations

Tsuji T
Department of Chemistry, Pennsylvania State University, University Park 16802.
Chrunyk BA
No affiliation info available
Chen X
No affiliation info available
Matthews CR
No affiliation info available

MeSH

Amino Acid SequenceBase SequenceCalorimetryCircular DichroismEnzyme StabilityEscherichia coliKineticsMacromolecular SubstancesMolecular Sequence DataMutagenesis, Site-DirectedOligodeoxyribonucleotidesPlasmidsProtein DenaturationProtein FoldingProtein Structure, SecondaryRecombinant ProteinsSalmonella typhimuriumTryptophan SynthaseUrea

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8504078

Citation

Tsuji, T, et al. "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." Biochemistry, vol. 32, no. 21, 1993, pp. 5566-75.
Tsuji T, Chrunyk BA, Chen X, et al. 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. Biochemistry. 1993;32(21):5566-75.
Tsuji, T., Chrunyk, B. A., Chen, X., & Matthews, C. R. (1993). 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. Biochemistry, 32(21), 5566-75.
Tsuji T, et al. 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. Biochemistry. 1993 Jun 1;32(21):5566-75. PubMed PMID: 8504078.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Tsuji,T, AU - Chrunyk,B A, AU - Chen,X, AU - Matthews,C R, PY - 1993/6/1/pubmed PY - 1993/6/1/medline PY - 1993/6/1/entrez SP - 5566 EP - 75 JF - Biochemistry JO - Biochemistry VL - 32 IS - 21 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8504078/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_ DB - PRIME DP - Unbound Medicine ER -