Dynamic structural changes of the photoactive orange carotenoid protein

• Applicant: Professor Dr. Thomas Friedrich
• Subject Area: Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics ; Biophysics ; Structural Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 379950877

The project aims to study the mechanisms of photoactivity of orange carotenoid protein (OCP). This pigment-protein complex is a sensor and effector of non-photochemical quenching (NPQ) in cyanobacteria and, because of its structural organization, it is a convenient model for the study of carotenoids, photoactive proteins and for the development of new biological trigger systems. The relevance is confirmed by recent high ranking publications in Scientific Reports, Plant Cell, PNAS, Science and other top-rated journals. In spite of the achievements of recent years, the structure of the red active form of the OCP is unknown, mostly due to inability to obtain stable crystals for X-ray structure analysis, as structural elements of OCP are characterized by high mobility. In this context, obtaining structural data and monitoring of the photochemical transformations of OCP will only be possible by using alternative structural methods: nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) in solution. To achieve these goals, we will use Escherichia coli strains that produce OCP from the cyanobacterium Synechocystis sp. PCC 6803 and provide a platform for the incorporation of proper carotenoid cofactors (beta-carotene and xanthophylls: echinenone, canthaxanthin, zeaxanthin). These carotenoid-producing E. coli strains are also necessary to obtain mutant forms of the protein to determine the role of specific amino acids in the process of photoconversion and to produce individual structural domains of OCP, which are important for initial stages of NMR experiments. Preparation of OCP proteins labeled with 13C and 15N isotopes will allow us to assign signals from 1H, 13C and 15N in NMR spectra and to determine a set of spectral parameters to calculate the OCP structure in solution. To study photocyclic transformations of OCP proteins, a comprehensive set of spectroscopic methods will be used: spectrofluorimetry with ps and fs time resolution, correlation spectroscopy, infrared and Raman spectroscopy, which will allow us to correlate structural data with the spectral characteristics of the protein. A combination of SAXS, SANS, and cross-linking mass-spectrometry (mapping interaction sites based on chemical "crosslinking" with subsequent mass spectrometric identification of "linked" peptides) will yield fundamental structural data on the interaction of the OCP with the Fluorescence Recovery Protein (FRP) protein, which interacts with OCP during NPQ cessation. The planned research will not only provide fundamental knowledge about regulatory mechanisms of NPQ in cyanobacteria, but also about the dynamics of conformational changes, in order to understand the principles of protein-protein and protein-chromophore interactions that determine its unique spectral characteristics. The results can be used to develop new protein sensor or photoswitch systems for biological systems research including optogenetics.

• DFG Programme: Research Grants
• International Connection: Russia
• Cooperation Partner: Dr. Eugene G. Maksimov, Ph.D.