Anti-Stokes fluorescence excitation reveals conformational mobility of the C-phycocyanin chromophores

Abstract

The structural organization of natural pigment-protein complexes provides a specific environment for the chromophore groups. Yet, proteins are inherently dynamic and conformationally mobile. In this work, we demonstrate the heterogeneity of chromophores of C-phycocyanin (C-PC) from Arthrospira platensis. Part of the population of trimeric C-PC is subject to spontaneous disturbances of protein–protein interactions resulting in increased conformational mobility of the chromophores. Upon fluorescence excitation in the visible range, the spectral signatures of these poorly populated states are masked by bulk chromophore states, but the former could be clearly discriminated when the fluorescence is excited by near-infrared quanta. Such selective excitation of conformationally mobile C-PC chromophores is due to the structure of their S1 level, which is characterized by a significantly broadened spectral line. We demonstrate that the anti-Stokes C-PC fluorescence is the result of single-photon absorption. By combining spectral and structural methods, we characterize four distinct states of C-PC chromophores emitting at 620, 650, 665, and 720 nm and assigned the fast component in the anti-Stokes fluorescence decay kinetics in the range of 690–750 nm to the chromophores with increased conformational mobility. Our data suggest that the spectral and temporal characteristics of the anti-Stokes fluorescence can be used to study protein dynamics and develop methods to visualize local environment parameters such as temperature.

Mirror proteorhodopsins

“…Small-angle X-ray scattering (SAXS) measurements with SpaR proteoliposomes were performed on SAXS instrument Rigaku MicroMax-007HF at MIPT (which previously was used and was described in works 132 – 134 ) to verify the formation of ULV. A standard model of the bilayer with a symmetric step EDP function was used for description of the SAXS scattering profile for liposomes.…”

luxAgene fromEnhygromyxa salinaencodes a functional homodimeric luciferase

“…The SAXS experiments were carried out on the Rigaku instrument at the Moscow Institute of Physics and Technology (Dolgoprudny, Russia) as described previously [47,48]. SAXS profiles were obtained for the peak fraction after gel-filtration with protein concentration of 3.5 mg/ml, in a buffer containing 150 mM NaCl and 25 mM Tris-HCl, pH 8.0.…”

“…The SAXS experiments were carried out on the Rigaku instrument at the Moscow Institute of Physics and Technology (Dolgoprudny, Russia) as described previously [48,49].…”

Recent Progress in Solution Structure Studies of Photosynthetic Proteins Using Small-Angle Scattering Methods

“…This is especially important when the oligomerization state of the protein complex under investigation is not known or deviates from the corresponding crystal structure. Examples include the oligomerization state of PC [68] and phycobiliproteins (PBP) from cyanobacteria [45] and the light-harvesting complexes LH1 and LH2 from purple bacteria [33] and LHCII from green plants [32].…”

“…In the case of phycocyanin, Tsoraev et al [68] showed using SAXS that the functional unit in their spectroscopic experiments was the PC trimer. Golub et al [45] employed SANS to study PBP from Acaryochloris marina in different buffer solutions yielding different functional competence and made use of the fact that this light-harvesting complex is composed of known components: one allophycocyanin trimer (APC) and an unknown number of PC trimers.…”