Bacteriorhodopsin Photocycle Captured at the Single Molecule Level by HS-AFM

Abstract

Bacteriorhodopsin (BR) from Halobacterium salinarum has been extensively studied structurally and functionally, and used as a model for light-driven proton pumps. Different techniques have been applied to study the structural intermediates and its dynamic response such as X-ray crystallography, X-ray free electron lasers (XFELs), solid-state NMR, high speed atomic force microscopy (HS-AFM) and more recently time-resolved serial femtosecond crystallography (TR-SFX). In particular, HS-AFM provided insights into the light-activation of BR studying cooperativity between protomers in neighboring trimers and revealed much larger conformational changes than anticipated of the surface exposed loops. In this work, we use two HS-AFM based approaches to characterize the photocycle of the molecule. We apply short light pulses and record HS-AFM line scanning HS-AFM (HS-AFM-LS) to determine the activation time response of the molecule. The activation time is much faster than reported before. Furthermore, under continuous light at different light intensities, we describe the full BR photocycle at the single molecule level and derive an energy landscape of the protein.