In broad terms the problem addressed is how biological signals are generated by a class of membrane proteins known as G-protein-coupled receptors (GPCRs). Rhodopsin is the major GPCR involved in vision, and serves as an archetype for determining molecular movies of receptors in action. Research will investigate the light activation mechanism of visual rhodopsin extracted from the retinal rod disk membranes into detergent micelles or small lipid nanodiscs. To monitor the protein shape and motion, the project will apply powerful new X-ray free electron laser (XFEL) technology. The goal is to study how light affects the protein shape and internal motions of rhodopsin over a range of different time scales. The significance and impact are twofold. First, it will illuminate the earliest events and photophysics of light activation process that occur in our eyes, together with the protein changes that yield vision. Second, it will showcase and drive the application of the new XFEL technology to membrane proteins that are not amenable to standard crystallization approaches. The primary benefit is to understand how light absorption by visual rhodopsin leads to changes in its mobility followed by transmission of a nerve signal to the brain. Time-resolved X-ray studies of rhodopsin in detergent solutions will reveal the protein motions triggered by light absorption of its cofactor (retinal, a derivative of Vitamin A). Computer simulations will further interpret the experimental observations in terms of changes in the dynamics of the protein molecules due to light. Important broader outcomes include training of biophysical scientists at the postdoctoral, graduate student, undergraduate, and high school levels, as well as teachers who will influence our society over many years to come.
The goals and scope of the research involve time-resolved X-ray scattering studies of the light activation of rhodopsin in detergent solutions and lipid nanodiscs. Changes in the structural dynamics of rhodopsin due to its photoactivation will be established over multiple scales of time and space: from sub picoseconds (10 C12 s) up to milliseconds (10 C3 s), and from chemical bond lengths up to entire protein molecules. Visual light from an optical parametric amplifier (OPA) will be used as the pump, with the short intense X-ray pulses of an XFEL as the probe. Pump-probe studies will establish how the ultrafast conformational changes of rhodopsin are propagated by a multiscale mechanism into the activated state of the receptor. The cofactor-induced changes in protein dynamics in the Photorhodopsin and Bathorhodopsin intermediates will be probed from the picosecond up to the nanosecond time scales to discover whether a "protein quake" occurs immediately after the initial light absorption. We will then study how the initial protein quake is propagated into the large-scale conformational fluctuations that activate rhodopsin. The Lumi and Meta-I states will be studied in the run-up to the active Meta-II state. We will discover how cis Ctrans isomerization of the retinal chromophore is focused to the dynamical hot spots of rhodopsin, which yield the activating conformational changes. Quantum mechanical/molecular dynamics (QM/MM) simulations will compare the theoretical difference-scattering profiles to experimental solution X-ray data to connect the light-induced changes to the atomic-resolution structure. Lastly, we will investigate binding of the C-terminal helix of the G-protein transducin in relation to the structural and dynamical alterations. Establishing how the ultrafast changes from light-induced isomerization of retinal are coupled to the large-scale protein fluctuations will greatly improve our understanding of rhodopsin activation and its role in visual signaling.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
