A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Abstract

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P fr , the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R C and P fr . In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

“…Previous studies with Agp1 indicated that mobility of ring A is required for photoconversion as follows: Agp1 assembled with synthetic chromophores in which the methine bridge between rings A and B is arrested in Za or Zs stereochemistry photoconverted either to a blueshifted adduct or to a species with unchanged absorbance maximum (52). Moreover, Agp1 with a fluorescein label at the position of the chromophore-binding site shows that the local environment of ring A changes significantly upon photoconversion (53).…”

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion

“…Three native cysteines, Cys-20, Cys-279, and Cys-295, all of them are potential binding sites for MTSL, can be found in the sequence of wild-type Agp1 phytochrome. Cys-279 and Cys-295 are deeply buried in the protein and not easily accessible for various labels (28). Nevertheless, they were successfully replaced by other amino acids without affecting the spectral properties or the enzymatic function of phytochrome.…”

“…Depending on the sample, we have observed varying efficiency of chromophore binding with typical values of 65-80% for the mutants presented here. Consequently, unoccupied Cys-20 could be accessible for other re-agents (28). Therefore, both the introduced cysteine and also to some extent Cys-20 were available for covalent linkage of cysteine-specific MTSL in each of the samples.…”

Light‐Induced Activation of Bacterial Phytochrome Agp1 Monitored by Static and Time‐Resolved FTIR Spectroscopy

“…[43] This interpretation is consistent with conclusions derived from the UV/Vis spectra of Agp1 adducts with conformationally locked biliverdin (BV) chromophores, CD spectroscopy and measurements with a polarity probe. [16,41,[44][45][46][47][48] To identify further localised structural changes of the chromophore, we take into account the results of the DFT calculations. Although these calculations refer to the cofactor in vacuo and thus are associated with intrinsic uncertainties, the calculated spectra help in identifying the nature of the characteristic difference band pairs below 1270 cm À1 in the Pfr/Pr difference spectrum.…”