文摘
Visual pigments have a conserved phenylalanine in transmembrane helix 5 located near the 尾-ionone ring of the retinal chromophore. Site-directed mutants of this residue (F207) in a short-wavelength sensitive visual pigment (VCOP) were studied using UV鈥搗isible spectroscopy to investigate its role in photosensitivity and formation of the light-activated state. The side chain is important for pigment formation: VCOP<sup>F207Asup>, VCOP<sup>F207Lsup>, VCOP<sup>F207Msup>, and VCOP<sup>F207Wsup> substitutions all bound 11-cis-retinal and formed a stable visual pigment, while VCOP<sup>F207Vsup>, VCOP<sup>F207Ssup>, VCOP<sup>F207Tsup>, and VCOP<sup>F207Ysup> substitutions do not. The extinction coefficients of all pigments are close, ranging between 35800 and 45600 M<sup>鈥?sup> cm<sup>鈥?sup>. Remarkably, the mutants exhibit an up to 5-fold reduction in photosensitivity and also abnormal photobleaching behavior. One mutant, VCOP<sup>F207Asup>, forms an isomeric composition of the retinal chromophore after illumination comparable to that of wild-type VCOP yet does not release the all-trans-retinal chromophore. These findings suggest that the conserved F207 residue is important for a normal photoactivation pathway, formation of the active conformation and the exit of all-trans-retinal from the chromophore-binding pocket.