Shixuan Liu1, Shuang Li1, Yihu Yang1, Weikai Li2. 1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. 2. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. weikai@wustl.edu.
Abstract
Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.
Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.
Authors: Maxime Killer; Giada Finocchio; Haydyn D T Mertens; Dmitri I Svergun; Els Pardon; Jan Steyaert; Christian Löw Journal: Front Mol Biosci Date: 2022-07-11
Authors: Sheng-Yi Wu; Yurong Wen; Nelson B C Serre; Cathrine Charlotte Heiede Laursen; Andrea Grostøl Dietz; Brian R Taylor; Mikhail Drobizhev; Rosana S Molina; Abhi Aggarwal; Vladimir Rancic; Michael Becker; Klaus Ballanyi; Kaspar Podgorski; Hajime Hirase; Maiken Nedergaard; Matyáš Fendrych; M Joanne Lemieux; Daniel F Eberl; Alan R Kay; Robert E Campbell; Yi Shen Journal: PLoS Biol Date: 2022-09-06 Impact factor: 9.593