Abstract

Purple membranes (PM) from *Halobacterium salinarum* serve as a critical model system for studying light-driven proton pumps, primarily due to the presence of the retinal-containing protein bacteriorhodopsin (bR). The functional photocycle of bR is intrinsically linked to subtle, yet crucial, structural rearrangements within the membrane lattice, which are challenging to resolve in real-time. This study investigates the applicability of Small-Angle X-ray Scattering (SAXS) as a non-invasive, high-throughput method to detect and characterize these illumination-driven structural changes in the PM. We employed time-resolved SAXS measurements on oriented PM samples under controlled light exposure to capture transient conformational states. The results demonstrate that SAXS is sufficiently sensitive to resolve changes in the inter-trimer lattice spacing and membrane thickness upon photoactivation, providing structural evidence for the dynamic nature of the bR photocycle. This successful application validates SAXS as a powerful tool for monitoring light-induced dynamics in two-dimensional membrane protein crystals, offering new avenues for understanding the structure-function relationship of membrane proteins in their native-like environment.