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The molecular structure and topography of the phytochrome chromophore
Abstract
Structural changes involved in the Pr $\to$ Pfr photoconversion of phytochrome were studied using surface-enhanced resonance Raman spectroscopy (SERRS). SERRS spectra of native 124 kDa phytochrome, chymotrypsin digested phytochrome and C-phycocyanin chromopeptides were measured. The SERRS studies indicate a Z $\to$ E isomerization occurs at the C$\sb{15}$-C$\sb{16}$ double bond during the Pr to Pfr photoconversion of phytochrome in the native protein. SERRS studies using deuterated sols indicate that the phytochrome chromophore is protonated in the Pr form, perhaps in the Pfr form. SERRS studies of the first photointermediate of the Pr $\to$ Pfr phototransformation tentatively suggest that the formation of Lumi-R involves an out of plane bending of ring-D along with a deprotonation and/or a breaking of hydrogen bonds to the chromophore. The reorientation of the phytochrome upon Pr $\to$ Pfr phototransformation was studied by Forster energy transfer from N-terminal specific fluorescently labeled Fab antibody fragments. The Forster energy transfer measurements indicate a chromophore reorientation during the phototransformation and a decrease in the distance between the N-terminus and the phytochrome chromophore of 10-13 A. The use of fluorescently labeled Fab fragments for Forster energy-transfer studies is a novel method of measuring distances within proteins. The pulsed-diode laser based time-correlated single-photon counting (TCSPC) system developed to perform these studies represents a low-cost alternative to standard laser based TCSPC systems.
Subject Area
Biochemistry|Biophysics
Recommended Citation
Farrens, David Lynn, "The molecular structure and topography of the phytochrome chromophore" (1991). ETD collection for University of Nebraska-Lincoln. AAI9219370.
https://digitalcommons.unl.edu/dissertations/AAI9219370