8DHR

An ester mutant of SfGFP

8DHR の概要

• エントリーDOI: 10.2210/pdb8dhr/pdb
• 分子名称: Green fluorescent protein (2 entities in total)
• 機能のキーワード: beta barrel, ester mutant, fluorescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 55546.39

構造登録者

Reddi, R.,Valiyaveetil, F.I. (登録日: 2022-06-28, 公開日: 2024-01-17)

主引用文献

Reddi, R.,Chatterjee, S.,Matulef, K.,Gustafson, A.,Gao, L.,Valiyaveetil, F.I.
A facile approach for incorporating tyrosine esters to probe ion-binding sites and backbone hydrogen bonds.
J.Biol.Chem., 300:105517-105517, 2023

PubMed Abstract: Amide-to-ester substitutions are used to study the role of the amide bonds of the protein backbone in protein structure, function, and folding. An amber suppressor tRNA/synthetase pair has been reported for incorporation of p-hydroxy-phenyl-L-lactic acid (HPLA), thereby introducing ester substitution at tyrosine residues. However, the application of this approach was limited due to the low yields of the modified proteins and the high cost of HPLA. Here we report the in vivo generation of HPLA from the significantly cheaper phenyl-L-lactic acid. We also construct an optimized plasmid with the HPLA suppressor tRNA/synthetase pair that provides higher yields of the modified proteins. The combination of the new plasmid and the in-situ generation of HPLA provides a facile and economical approach for introducing tyrosine ester substitutions. We demonstrate the utility of this approach by introducing tyrosine ester substitutions into the K channel KcsA and the integral membrane enzyme GlpG. We introduce the tyrosine ester in the selectivity filter of the M96V mutant of the KcsA to probe the role of the second ion binding site in the conformation of the selectivity filter and the process of inactivation. We use tyrosine ester substitutions in GlpG to perturb backbone H-bonds to investigate the contribution of these H-bonds to membrane protein stability. We anticipate that the approach developed in this study will facilitate further investigations using tyrosine ester substitutions.

PubMed: 38042487
DOI: 10.1016/j.jbc.2023.105517

実験手法

X-RAY DIFFRACTION (1.75 Å)