1L39

CONTRIBUTIONS OF ENGINEERED SURFACE SALT BRIDGES TO THE STABILITY OF T4 LYSOZYME DETERMINED BY DIRECTED MUTAGENESIS

1L39 の概要

• エントリーDOI: 10.2210/pdb1l39/pdb
• 分子名称: T4 LYSOZYME (2 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: 18643.38

構造登録者

Daopin, S.,Matthews, B.W. (登録日: 1991-01-28, 公開日: 1991-10-15, 最終更新日: 2024-05-22)

主引用文献

Sun, D.P.,Sauer, U.,Nicholson, H.,Matthews, B.W.
Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.
Biochemistry, 30:7142-7153, 1991

PubMed Abstract: Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.

PubMed: 1854726
DOI: 10.1021/bi00243a015

実験手法

X-RAY DIFFRACTION (1.85 Å)