1PQO

T4 Lysozyme Core Repacking Mutant L118I/TA

1PQO の概要

• エントリーDOI: 10.2210/pdb1pqo/pdb
• 関連するPDBエントリー: 1P2L 1P2R 1P36 1P37 1P3N 1P64 1P6Y 1P7S 1PQD 1PQI 1PQJ 1PQM
• 分子名称: Lysozyme, POTASSIUM ION, CHLORIDE ION, ... (5 entities in total)
• 機能のキーワード: hydrolase (o-glycosyl), t4 lysozyme, designed core mutant, automated protein design, protein engineering, protein folding, protein stability, core repacking, back revertant, dead-end elimination theorem, side-chain packing, optimized rotamer combinations, orbit, hydrolase
• 由来する生物種: Enterobacteria phage T4
• 細胞内の位置: Host cytoplasm : P00720
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 18892.62

構造登録者

Mooers, B.H.,Datta, D.,Baase, W.A.,Zollars, E.S.,Mayo, S.L.,Matthews, B.W. (登録日: 2003-06-18, 公開日: 2003-10-07, 最終更新日: 2023-08-16)

主引用文献

Mooers, B.H.,Datta, D.,Baase, W.A.,Zollars, E.S.,Mayo, S.L.,Matthews, B.W.
Repacking the Core of T4 lysozyme by automated design
J.Mol.Biol., 332:741-756, 2003

PubMed Abstract: Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

PubMed: 12963380
DOI: 10.1016/S0022-2836(03)00856-8

実験手法

X-RAY DIFFRACTION (1.65 Å)