Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Pea phytochrome A: Chromophore attachment and photoreversibility
Abstract
Photomorphogenesis in plants is controlled, in part, by the phytochromes, a family of light receptors that are reversibly interconvertible between a red light absorbing form (P$\sb{\rm r}$) and a far-red light absorbing form (P$\sb{\rm fr}$). Phytochrome is synthesized as the former, while the latter triggers photomorphogenesis. Pea phytochrome A is shown to be a predominantly $\alpha$-helical protein which undergoes a conformational change when phototransformed from P$\sb{\rm r}$ to P$\sb{\rm fr}$. The percentage of residues which adopt the $\alpha$-helical conformation increases with 1.5 to 5% when P$\sb{\rm fr}$ is formed and these conformational changes likely take place in the N-terminal domain of the molecule. The gene for pea phytochrome A was expressed in yeast and the apophytochrome is shown to assemble in vitro with phycocyanobilin to yield a photoreversible holophytochrome. Three pea phytochrome A deletion mutants were analyzed. A deletion of the N-terminal domain to residue 46 does not affect chromophore attachment and photoreversibility. However, when 222 amino acids are deleted, holophytochrome cannot be assembled in vitro under the same conditions. A truncation of the entire C-terminal half of the phytochrome molecule still allows chromophore attachment and photoreversibility. This indicates that the catalytic site for phytochrome lyase activity resides in the N-terminal half of the protein. The hydrophobic chromophore pocket of all phytochromes is very well conserved. A mutational analysis of specific amino acids, which are located adjacent to the chromophore attachment site, was carried out. Ten mutants were generated, comprising two changes for each of five conserved amino acids. All mutants attach the chromophore covalently in vitro but the rate of chromophore incorporation varies considerably. The mutant holophytochromes are spectrally of three types: mutants which are wild-type like, mutants which exhibit blue-shifted absorption maxima compared to the wild-type adduct, and mutants in which the red/far-red photoreversibility is abolished. This research represents a first experimental probing for specific bilin/protein interactions in the phytochrome chromophore pocket and allows to elaborate on current phytochrome models.
Subject Area
Biochemistry
Recommended Citation
Deforce, Lily Marie, "Pea phytochrome A: Chromophore attachment and photoreversibility" (1993). ETD collection for University of Nebraska-Lincoln. AAI9333961.
https://digitalcommons.unl.edu/dissertations/AAI9333961