Sensory Rhodopsin II (SRII) is a photo-active integral membrane protein with seven transmembrane α-helices and a covalently bound retinal chromophore. Activation with near UV-blue light isomerizes the all-trans chromophore to a 13-cis conformation, which initiates a negative phototaxis response. SRII binds in the membrane to a transducer protein (HtrII) through which it transmits a structural signal. The aim of this work is to study the structural changes in SRII and understand how the signal propagates from SRII to its complexed transducer protein.
Time-resolved crystallography (TRX) allows protein conformational changes to be tracked with time at atomic resolution. Time-resolved Serial Millisecond Crystallography (TR-SMX) data were collected from microcrystals of SRII at the PX1 beamline of the Swiss Light Source (SLS). A steady state difference electron density map for SRII was calculated to 2.1Å diffraction. Overall, helices C and F showed clear movements, but a movement of helix-G that was previously observed for bacteriorhodopsin1 (bR) was not present. This finding is explained by hydrogen bond interactions of Thr204 increasing the rigidity of helix-G of SRII relative to bR.
Time-resolved X-ray solution scattering (TR-XSS) was used to study conformational changes in SRII and in the SRII:HtrII complex from microseconds to milliseconds. TR-XSS provides low-resolution structural information about secondary structure rearrangements in proteins and these motions are not constricted by a crystal lattice. Scattering data were recorded at ID09B of the ESRF for both SRII in isolation and when bound to HtrII. Preliminary analyses of time-dependent changes in X-ray scattering show similar movements to those previously observed for bR using TR-XSS but with the amplitude of helix motions being reduced2 for the SRII:HtrII complex. Further improvements in our routines for fitting TR-XSS data are expected to provide improved structural fits to these data and thereby reveal different conformations over time.