The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L.

Abstract

The spectral properties of peptides generated from etiolated-Avana, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 nm, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structureal integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722-724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6-10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 63-kDa species leads to aberrant spectral properties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/114-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule.