DIFFERENTIAL EXPOSURE OF TRYPTOPHAN RESIDUES IN THE RED AND FAR-RED LIGHT ABSORBING FORMS OF PHYTOCHROME, AS REVEALED BY CHEMICAL MODIFICATION

The effects of the chemical modification of tryptophan residues in native pea (Pisum sativum L.) phytochrome by 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) were examined. Such treatment had no effect on the spectral properties or on the pattern of tryptic digestion of phytochrome, which indicated that no major conformational change in phytochrome had occurred. Amino acid analysis of the HNB-Br-treated phytochrome indicated that the number of modified Trp residues after the treatment was dependent on the light-absorbing form. The values were three for P-R and five for P-FR (out of a total of ten) per monomer. The results indicate that two additional Trp residues are exposed on the molecular surface of P-FR when the photoconversion of P-R to P-FR occurs. The amino acid analysis of a 58-kDa tryptic fragment of phytochrome (a mixture of peptides, residues 63-583 and 66-587) showed that one Trp residue in the fragment from P-R and two in that from P-FR (out of six) were modified by HNB-Br. In the 56-kDa fragment (a mixture of peptides, residues 598-1121 and 603-1124), there were two modified Trp residues in P-R and three in P-FR (out of four). The Trp residue in a 36-kDa fragment (residues 66-383), which includes the tetrapyrrolic chromophore, was not modified in the either case. These results indicate that new exposed sites that are generated by the photoconversion of P-R to P-FR are in the region between Trp-456 and Trp-567 and in that between Trp-644 and Trp-787.