FUNCTIONAL EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF PHOTOACTIVE YELLOW PROTEIN

The gene encoding photoactive yellow protein (PYP) was isolated from E ctothiorhodospira halophila, and a high-level expression system for PY P was constructed in Escherichia coil, The molecular weight and the ab sorption spectrum of PYP expressed in E, coil were identical with thos e of the native PYP isolated from E, halophila, The amino acid residue s which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg5 2, and Cys69) were mutated by site-directed mutagenesis and the absorp tion spectra of these mutants were examined to study the chromophore/p rotein interaction in PYP,The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments, These results suggest that Ty r42, Glu46, and Thr50 strongly interact with the chromophore, while Ar g52 does not contribute the color tuning of PYP.