Optimization of the gbeta1 domain by computational design and by in vitro evolution: structural and energetic basis of stabilization |
| |
Authors: | Wunderlich Michael Max Klaas E A Roske Yvette Mueller Uwe Heinemann Udo Schmid Franz X |
| |
Affiliation: | Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universit?t Bayreuth, D-95440 Bayreuth, Germany. |
| |
Abstract: | Computational design and in vitro evolution are major strategies for stabilizing proteins. For the four critical positions 16, 18, 25, and 29 of the B domain of the streptococcal protein G (Gbeta1), they identified the same optimal residues at positions 16 and 25, but not at 18 and 29. Here we analyzed the energetic contributions of the residues from these two approaches by single and double mutant analyses and determined crystal structures for a variant from the calculation (I16/L18/E25/K29) and from the selection (I16/I18/E25/F29). The structural analysis explains the observed differences in stabilization. Residues 16, 18, and 29 line an invagination, which results from a packing defect between the helix and the beta-sheet of Gbeta1. In all stabilized variants, residues with larger side-chains occur at these positions and packing is improved. In the selected variant, packing is better optimized than in the computed variant. Such differences in side-chain packing strongly affect stability but are difficult to evaluate by computation. |
| |
Keywords: | Gβ1, β1 domain of the streptococcal protein G Gβ1-ILEK, a variant with the mutations T16I, T18L, T25E, and V29K (Gβ1c3-ILEK contains also the mutations Y3F, L7I, and V39I) Gβ1-IIEF, a variant with the mutations T16I, T18I, T25E, and V29F (Gβ1c3-IIEF contains also the mutations Y3F, L7I, and V39I) G3P, gene-3-protein of phage fd TM, midpoint of a thermal unfolding transition GdmCl, guanidinium chloride [GdmCl]m, midpoint of a GdmCl-induced unfolding transition m, cooperativity parameter of a GdmCl-induced unfolding transition, m = ∂ΔGD/∂[GdmCl] |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|