Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformations

Abstract

Phytochrome was examined by immunochemical and spectroscopic techniques to detect differences between the protein moieties of red- and far red-absorbing phytochrome (P(r) and P(fr)). No differences in the reaction of P(r) and P(fr) with phytochrome antibody were discernible on Ouchterlony double diffusion plates. However, the microcomplement fixation assay showed a greater degree of antibody reaction with P(fr) than with P(r), indicating some difference in the surface characteristics of the two forms. Circular dichroism spectroscopy between 300 and 200 nanometers revealed differences between P(r) and P(fr) which may reflect differences in the protein conformation. The circular dichroism spectrum of P(r) showed a negative band at 285 nanometers which was not present in the spectrum of P(fr), and the large negative circular dichroism band at 222 nanometers with P(fr), associated with the alpha-helical content, was shifted 2 nanometers to shorter wave length with P(r) although there was no change of magnitude of this band. The absorbancy of P(r) and P(fr) is very nearly the same in the 280 nanometer spectral region, but sensitive difference spectra between P(r) and P(fr) did reveal spectra which were similar to solvent perturbation spectra obtained by others with different proteins. In total, the experiments indicate that there are conformational differences between the protein moieties of P(r) and P(fr) but that these differences are rather slight from a standpoint of gross structure.