A CONFORMATIONAL CHANGE ASSOCIATED WITH THE PHOTOTRANSFORMATION OF PISUM PHYTOCHROME-A AS PROBED BY FLUORESCENCE QUENCHING

Dynamic quenching of the two lifetime component tryptophan fluorescenc e of Pisum phytochrome has revealed differential accessibility of cert ain residues. Both acrylamide and Tl+ ions showed preferential exposur e of some tryptophans in Pfr-phytochrome. Greater k(q)'s for Pfr are, however, in contrast with values for Avena phytochrome in which Pr --> Pfr conversion impedes Tl+ access. The Pr short lifetime component wa s more accessible to Cs+; however, the long component accessibility wa s approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce t he total number of fluorescent tryptophans. The absence of the fluores cence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ K(sv)'s for Pr and Pfr. The two a dditional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The reg ions around four of the 10 tryptophans may represent conformationally photoresponsive areas in Pisum phytochrome A. Furthermore, topographic changes associated with the phytochrome phototransformation are not c onfined to the 58-kDa chromphore domain, and they involve most if not all of the region from Trp-365 to Trp-787. We also provide evidence th at the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.