3DGS

Changing the determinants of protein stability from covalent to non-covalent interactions by in-vitro evolution: a structural and energetic analysis

3DGS の概要

• エントリーDOI: 10.2210/pdb3dgs/pdb
• 分子名称: Coat protein A (2 entities in total)
• 機能のキーワード: protein stabilization, dissulfide bonds, evolutionary protein design, phage gene-3-protein, phage display, capsid protein, phage recognition, virion, viral protein
• 由来する生物種: Bacteriophage fd
• 細胞内の位置: Virion (Potential): P03661
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 49551.92

構造登録者

Jakob, R.P.,Kather, I.,Dobbek, H.,Schmid, F.X. (登録日: 2008-06-16, 公開日: 2008-07-22, 最終更新日: 2023-11-01)

主引用文献

Kather, I.,Jakob, R.P.,Dobbek, H.,Schmid, F.X.
Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis.
J.Mol.Biol., 381:1040-1054, 2008

PubMed Abstract: The three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol(-1). This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability.

PubMed: 18621056
DOI: 10.1016/j.jmb.2008.06.073

実験手法

X-RAY DIFFRACTION (1.9 Å)