Time-resolved absorption and photothermal measurements with recombinant sensory rhodopsin II from Natronobacterium pharaonis

Purified wild-type sensory rhodopsin II from Natronobacterium pharaonis (pS RII-WT) and its histidine-tagged analog (pSRII-His) were studied by laser-i nduced optoacoustic spectroscopy (LIOAS) and flash photolysis with optical detection. The samples were either dissolved in detergent or reconstituted into polar lipids from purple membrane (PML). The quantum yield for the for mation of the long-lived state M-400 was determined as phi(M) = 0.5 +/- 0.0 6 for both proteins. The structural volume change accompanying the producti on of K-510 as determined with LIOAS was Delta V-R,V-1 less than or equal t o 10 ml for both proteins, assuming phi(K) greater than or equal to phi(M), indicating that the His tag does not influence this early step of the phot ocycle. The medium has no influence on Delta V-R,V-1 which is the largest s o far measured for a retinal protein in this time range (< 10 ns). This con firms the occurrence of conformational movements in pSRII for this step, as previously suggested by Fourier transform infrared spectroscopy. On the co ntrary, the decay of K-510 is an expansion in the detergent-dissolved sampl e and a contraction in PML. Assuming an efficiency of 1.0, Delta V-R,V-2 = -3 ml/mol for pSRII-WT and -4.6 ml/mol for pSRII-His were calculated in PML , indicative of a small structural difference between the two proteins. The energy content of K-510 is also affected by the tag. It is E-K = (88 +/- 1 3) for pSRII-WT and (134 +/- 11) kJ/mol for pSRII-His. A slight difference in the activation parameters for K-510 decay confirms an influence of the C -terminal His on this step. At variance with Delta V-R,V-1 the opposite sig n of Delta V-R,V-2 in detergent and PML suggests the occurrence of solvatio n effects on the decay of K-510, which are probably due to a different inte raction of the active site with the two dissolving media.