Atomic Force Spectroscopy Measures Light Activation And Transducer Binding Induced Structural Changes In The Sensory Rhodopsin II

Abstract

Rhodopsins are a family of seven-helical transmembrane proteins responding to light. Sensory rhodopsin II (SRII) triggers two very different responses depending on the presence or absence of its transducer: Whereas light activation of the SRII-transducer-complex triggers a signalling cascade initiating the photophobic response of the bacterium, SRII alone acts as a proton-pump.Using single molecule force spectroscopy we analysed the stability of SRII in dark and after light activation as well as in presence and absence of the transducer, which revealed a distinct pattern of changes in the protein stability. By improving the force spectroscopic data analysis we were able to predict the localisation of occuring forces within the protein chain with a resolution of about six amino acids.Different regions showed up, where secondary structure elements of SRII are selectively stabilised or weakened by either light activation or transducer binding or both. Independent of the presence of the transducer light activation has a destabilizing effect in the middle of α-helix G. This suggests a loss in interactions between helices G and F, which would allow an outward tilt of α-helix F as previously observed. Additionally, the unfolding curves show an increased number of rupture events in the region of helix F upon transducer binding, which is most likely due to the formation of several interactions between α-helix F and TM2 of the transducer. Most interestingly, we found a loss of some of these interactions upon light activation, which might explain transducer activation and help to answer remaining questions concerning the precise molecular mechanism. Finally, in the absence of the transducer, destabilizing effects are observed at the cytoplasmic half of helix G, which might indicate its importance for the proton-pumping properties of SRII.