Structural Dynamics of Light-Driven Proton Pumps

Abstract

Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

“…Time-resolved wide-angle X-ray scattering has also recently been extended to probe the conformational dynamics of proteins Andersson et al, 2009). This approach can be viewed either as an extension of the small molecule studies above to more complex systems, or as a higher-resolution extension of the method of time-resolved small-angle X-ray scattering, which provides low-resolution structural information on the dynamics of protein and RNA folding and oligomeric assembly (Lamb, Kwok et al, 2008;Lamb, Zoltowski et al, 2008;Wu et al, 2008;Canady et al, 2001;Russell et al, 2000;Segel et al, 1999).…”

“…Unlike haemoglobin, however, the nature, extent and timing of secondary structural element rearrangements within the photocycle of bacteriorhodopsin have been controversial (Hirai & Subramaniam, 2009;Andersson et al, 2009). Fig.…”

“…Significant technical challenges will also be encountered when moving from light-driven and reversible proof-ofprinciple systems of study Andersson et al, 2009) to structurally characterize the conformational dynamics of chemically driven irreversible reactions, but there are no reasons in principle why these challenges cannot be overcome. Promising options in this respect include developing micro-fluidics stopped-flow devices or the use of caged compounds to allow light activation of chemically driven reactions, with the latter technology being particularly well developed in other fields of biology (Ellis-Davies, 2007;Kao, 2006;Mayer & Heckel, 2006).…”

Application of Stopped-Flow and Time-Resolved X-Ray Absorption Spectroscopy to the Study of Metalloproteins Molecular Mechanisms

“…laser-flash excitation) for initiating the reaction and by varying the delay time between the trigger and X-ray pulse, one can determine individual structures separated by time intervals of picoseconds [92][93][94] . Time-resolved wide-angle X-ray scattering (TR-WAXS) accurately probes tertiary and quaternary structural changes in proteins in solution with nanosecond time resolution 95 . In a TR-WAXS experiment, a laser pulse is used to trigger the protein's structural change, and transient structures are then www.intechopen.com…”

SAXS-Guided Metadynamics

“…[10][11] Membrane protein targets seem conceivable considering nanodisc technology 12 and modeling of surrounding lipids/detergents. 13 In addition, time-resolved WAXS (TR-WAXS) of soluble proteins [14][15] and membrane proteins [16][17] show great promise for adaptation to the X-ray free electron laser (XFEL) technology. [18][19] The molecules in the sample are not oriented in space and the diffraction pattern obtained will therefore represent a rotational average in reciprocal space corresponding to the multitude of displayed molecular orientations.…”