A Conformational Change Associated with the Phototransformation of Pisum Phytochrome A As Probed by Fluorescence Quenching

Abstract

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.

Phytochrome Phosphorylation Modulates Light Signaling by Influencing the Protein–Protein Interaction[W]

“…On Pr/Pfr phototransformation, the NTE region undergoes a conformational change from random coil to amphiphilic a-helix, which then interacts with the chromophore in the Pfr form (Parker et al, 1992;Deforce et al, 1994). Also, two Trp residues near the core regulatory region of oat phyA become preferentially exposed in the Pfr form (Wells et al, 1994). Based on these results, it was proposed that part of the NTE chain interacts with the regulatory C-terminal core motif in the Pr form (switch off conformation) (Park et al, 2000;Kim et al, 2002b).…”

Reaction Mechanism of Plant 2-Cys Peroxiredoxin

“…7 shows the fluorescence lifetimes determined for Prx WT and its Trp variants under oxidizing and reducing conditions. With exception of the mutant W189L, which revealed typical lifetimes for Trp within a protein (37)(38)(39), the averaged fluorescence lifetimes of all samples investigated were significantly lower than those of tryptophan in aqueous solution ( m ϭ 2610 ps, 1 ϭ 2940 ps, 2 ϭ 280 ps; determined at emission maximum em ϭ 350 nm) (Table II). This means that the lifetimes of both fluorophores were significantly shorter than those of free, solvent-accessible tryptophans, although they are shielded in the protein matrix (which usually leads to longer fluorescence lifetimes).…”

Chromophore: apoprotein interactions in phytochrome A*

“…During phytochrome phototransformation, Tip-365 appears to remain buried, whereas Tip-456, -473, -567, -773 and -777 in pea phyA become exposed preferentially in the Pfr form (Wells et al 1994). This result may also reflect an inter-domain interaction between the chromophore/ chromophore peptide and C-temiinal domains.…”

“…Also, the buried a-helical motif in residues 360-400 of pea phyA appears to play a critical role in the chromophore-apoprotein interactions involved in the photochromism of phyA. The Trp-365 residue of pea phyA remains buried during Pr to Pfr phototransformation, partly contributing to the hydrophobie pocket for the chromophore (Wells et al 1994). Other peptide segments within the N-terminal domain of the first 600 amino acid residues seem to play only a minor role in determining the spectral maxima of the Pr and Pfr forms of phyA.…”