Infrared nanospectroscopy study of the light-induced conformational changes of Channelrhodopsin

Maria Eleonora Temperini,Ulrich Schade,Eglof Ritter,Michele Ortolani,Valeria Giliberti,Leonetta Baldassarre,Raffaella Polito,Ljljana Puskar,Antonia Intze,C Sibilia,G Pauliat,A Belardini

doi:10.1051/epjconf/202125513001

Abstract

The channelrhodopsin-ChR2 is a light-sensitive transmembrane protein that acts as a selective ion channel between the intra- and the extra-cellular environments. In the last decade, ChR2 has proven to be essential for optogenetics, because, if expressed in mammalian neural cells, it enables the control of neuronal activity in response to visible light. Mid-infrared difference spectroscopy can probe the functional conformational changes of light-sensitive proteins, however intrinsic limitations of standard IR spectroscopy in terms of diffraction, and therefore number of probed proteins, require that the mid-IR experiments be performed on huge numbers of lipid membrane patches with overexpressed proteins. In this work, we apply for the first time IR difference nanospectroscopy, based on the use of mid-IR lasers and an atomic force microscope (AFM), to single membrane patches containing ChR2, obtaining relevant spectroscopy results for optogenetic applications and, more generally, for future experimental studies of light-sensitive proteins at the nanoscale.

Highlights

Light-sensitive microbial rhodopsins are proteins that play a crucial role in the cell functions acting as ion pumps or channels across the cell membrane in response to absorption of visible light
Mid-infrared difference spectroscopy can probe the functional conformational changes of light-sensitive proteins, intrinsic limitations of standard IR spectroscopy in terms of diffraction, and number of probed proteins, require that the mid-IR experiments be performed on huge numbers of lipid membrane patches with overexpressed proteins
We apply for the first time IR difference nanospectroscopy, based on the use of mid-IR lasers and an atomic force microscope (AFM), to single membrane patches containing ChR2, obtaining relevant spectroscopy results for optogenetic applications and, more generally, for future experimental studies of light-sensitive proteins at the nanoscale

Summary

Introduction

Light-sensitive microbial rhodopsins are proteins that play a crucial role in the cell functions acting as ion pumps or channels across the cell membrane in response to absorption of visible light. Mid-infrared difference spectroscopy can probe the functional conformational changes of light-sensitive proteins, intrinsic limitations of standard IR spectroscopy in terms of diffraction, and number of probed proteins, require that the mid-IR experiments be performed on huge numbers of lipid membrane patches with overexpressed proteins.

Results

Conclusion