Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Abstract

Significance Light-driven rhodopsin proteins pump ions across cell membranes. They have applications in optogenetics and can potentially be used to develop solar energy–harvesting devices. A detailed understanding of rhodopsin dynamics and functions may therefore assist research in medicine, health, and clean energy. This time-resolved crystallography study carried out with X-ray free-electron lasers reveals detailed dynamics of chloride ion–pumping rhodopsin (ClR) within 100 ps of light activation. It shows the dissociation of Cl− from the Schiff base binding site upon light-triggered retinal isomerization. This Cl− dissociation is followed by diffusion toward the intracellular direction. The results hint at a common ion-pumping mechanism across rhodopsin families. Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl− into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.