Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions

Albert Godoy-Hernandez; Amer H Asseri; Aiden J Purugganan; Chimari Jiko; Carol de Ram; Holger Lill; Martin Pabst; Kaoru Mitsuoka; Christoph Gerle; Dirk Bald; Duncan G G McMillan

doi:10.1021/acscentsci.2c01170

Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions

ACS Cent Sci. 2023 Feb 22;9(3):494-507. doi: 10.1021/acscentsci.2c01170. eCollection 2023 Mar 22.

Authors

Albert Godoy-Hernandez¹, Amer H Asseri^{2

3}, Aiden J Purugganan¹, Chimari Jiko⁴, Carol de Ram¹, Holger Lill³, Martin Pabst¹, Kaoru Mitsuoka⁵, Christoph Gerle^{6

7}, Dirk Bald³, Duncan G G McMillan^{1

8}

Affiliations

¹ Department of Biotechnology, Delft University of Technology, 2628 CD Delft, The Netherlands.
² Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
³ Amsterdam Institute for Life and Environment (A-LIFE), AIMMS, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
⁴ Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kyoto, 606-8501, Japan.
⁵ Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Ibaraki, Osaka 565-0871, Japan.
⁶ Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
⁷ Life Science Research Infrastructure Group, RIKEN SPring-8 Center, Kouto, Hyogo 679-5148, Japan.
⁸ Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Bunkyo City, Tokyo 113-8654, Japan.

Abstract

Functional reintegration into lipid environments represents a major challenge for in vitro investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability. LAiR allowed for monitoring catalysis of a membrane-bound, physiologically relevant polyisoprenoid quinone substrate by Escherichia coli cytochromes bo ₃ (cbo ₃) and bd (cbd) under control of the proton motive force. LAiR also facilitated bulk-phase detection and physiological assessment of the "proton leak" in cbo ₃, a controversial catalytic state that previously was only approachable at the single-molecule level. LAiR maintained the multisubunit integrity and higher-order oligomeric states of the delicate mammalian F-ATP synthase. Given that LAiR can be applied to both liposomes and planar membrane bilayers and is compatible with IMPs and lipids from prokaryotic and eukaryotic sources, we anticipate LAiR to be applied broadly across basic research, pharmaceutical applications, and biotechnology.