4LQU

1.60A resolution crystal structure of a superfolder green fluorescent protein (W57G) mutant

4LQU の概要

• エントリーDOI: 10.2210/pdb4lqu/pdb
• 関連するPDBエントリー: 4LQT
• 分子名称: Green fluorescent protein (2 entities in total)
• 機能のキーワード: gfp, chemical biology, allosteric activation, switchable enzyme, chemical rescue, mutation, fluorescent protein
• 由来する生物種: Aequorea victoria (Jellyfish)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 110736.15

構造登録者

Lovell, S.,Xia, Y.,Vo, B.,Battaile, K.P.,Egan, C.,Karanicolas, J. (登録日: 2013-07-19, 公開日: 2013-12-18, 最終更新日: 2024-11-27)

主引用文献

Xia, Y.,DiPrimio, N.,Keppel, T.R.,Vo, B.,Fraser, K.,Battaile, K.P.,Egan, C.,Bystroff, C.,Lovell, S.,Weis, D.D.,Anderson, J.C.,Karanicolas, J.
The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation.
J.Am.Chem.Soc., 135:18840-18849, 2013

PubMed Abstract: The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, X-ray crystallography, and hydrogen-deuterium mass spectrometry, we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.

PubMed: 24313858
DOI: 10.1021/ja407644b

実験手法

X-RAY DIFFRACTION (1.6 Å)