138L

RAPID CRYSTALLIZATION OF T4 LYSOZYME BY INTERMOLECULAR DISULFIDE CROSSLINKING

138L の概要

• エントリーDOI: 10.2210/pdb138l/pdb
• 分子名称: T4 LYSOZYME, CHLORIDE ION, BETA-MERCAPTOETHANOL, ... (4 entities in total)
• 機能のキーワード: hydrolase(o-glycosyl)
• 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: 18809.47

構造登録者

Heinz, D.W.,Matthews, B.W. (登録日: 1993-09-01, 公開日: 1994-01-31, 最終更新日: 2024-02-07)

主引用文献

Heinz, D.W.,Matthews, B.W.
Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking.
Protein Eng., 7:301-307, 1994

PubMed Abstract: In an attempt to facilitate crystallization, engineered cysteines were used to promote formation of a 'back-to-back' dimer that occurs in different crystal forms of wild-type and mutant T4 lysozymes. The designed double mutant, N68C/A93C, in which the surface residues Asn68 and Ala93 were replaced by cysteines, formed dimers in solution and crystallized isomorphously to wild-type, but at a much faster rate. Overall, the mutant structure remained very similar to wild-type despite the formation of two intermolecular disulfide bridges. The crystals of cross-linked dimers ahd thermal factors somewhat lower than wild-type, indicating reduced mobility, but did not diffract to noticeably higher resolution. Introduction of the same cross-links was also used to obtain crystals in a different space group of a T4 lysozyme mutant that could not be crystallized previously. The results suggest that the formation of the lysozyme dimer is a critical intermediate in the formation of more than one crystal form and that covalent cross-linking of the intermediate accelerates nucleation and facilitates crystal growth. The disulfide cross-links are located on the 'back' of the molecule and formation of the cross-linked dimer appears to leave the active sites completely unobstructed. Nevertheless, the cross-linked dimer is completely inactive. One explanation for this behavior is that the disulfide bridges prevent hinge-bending motion that may be required for catalysis. Another possibility is that the formation of the dimer increases the overall bulk of the enzyme and prevents its access to the susceptible glycosidic bonds within the cell wall substrate.

PubMed: 8177878
DOI: 10.1093/protein/7.3.301

実験手法

X-RAY DIFFRACTION (1.7 Å)