A conformational change associated with the phototransformation of Pisum phytochrome A as probed by fluorescence quenching.
Dynamic quenching of the two lifetime component tryptophan fluorescence of Pisum phytochrome has revealed differential accessibility of certain residues. Both acrylamide and Tl+ ions showed preferential exposure of some tryptophans in Pfr-phytochrome. Greater kq'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 was more accessible to Cs+; however, the long component accessibility was approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce the total number of fluorescent tryptophans. The absence of the fluorescence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ Ksv's for Pr and Pfr. The two additional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The regions 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 confined 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 that the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.
Wells
T A
TA
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Nakazawa
M
M
Manabe
K
K
Song
P S
PS
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000494:pharmacology; D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D002586:Cesium / Q000494:pharmacology; D007887:Fabaceae / Q000737:chemistry*; D008027:Light; D010834:Phytochrome / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects*; D010946:Plants, Medicinal; D013050:Spectrometry, Fluorescence; D013793:Thallium / Q000494:pharmacology; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D000178:Acrylamides; D010834:Phytochrome; D002586:Cesium; D020106:Acrylamide; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D013793:Thallium