Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems

Marcus Moldenhauer; Hsueh-Wei Tseng; Anastasia Kraskov; Neslihan N Tavraz; Igor A Yaroshevich; Peter Hildebrandt; Nikolai N Sluchanko; Georg A Hochberg; Lars-Oliver Essen; Nediljko Budisa; Lukas Korf; Eugene G Maksimov; Thomas Friedrich

doi:10.3389/fmolb.2023.1072606

Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems

Front Mol Biosci. 2023 Jan 26:10:1072606. doi: 10.3389/fmolb.2023.1072606. eCollection 2023.

Affiliations

¹ Department of Bioenergetics, Institute of Chemistry PC 14, Technische Universität Berlin, Berlin, Germany.
² Department of Biocatalysis, Institute of Chemistry L1, Technische Universität Berlin, Berlin, Germany.
³ Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia.
⁴ A.N. Bach Institute of Biochemistry, Federal Research Center Fundamentals of Biotechnology of Russian Academy of Sciences, Moscow, Russia.
⁵ Max-Planck-Institute of Terrestrial Microbiology, Evolutionary Biochemistry Group, Marburg, Germany.
⁶ Department of Chemistry and Unit for Structural Biology, Philipps-Universität Marburg, Marburg, Germany.
⁷ Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada.

Abstract

Introduction: Dissecting the intricate networks of covalent and non-covalent interactions that stabilize complex protein structures is notoriously difficult and requires subtle atomic-level exchanges to precisely affect local chemical functionality. The function of the Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, depends strongly on two H-bonds between the 4-ketolated xanthophyll cofactor and two highly conserved residues in the C-terminal domain (Trp288 and Tyr201). Method: By orthogonal translation, we replaced Trp288 in Synechocystis OCP with 3-benzothienyl-L-alanine (BTA), thereby exchanging the imino nitrogen for a sulphur atom. Results: Although the high-resolution (1.8 Å) crystal structure of the fully photoactive OCP-W288_BTA protein showed perfect isomorphism to the native structure, the spectroscopic and kinetic properties changed distinctly. We accurately parameterized the effects of the absence of a single H-bond on the spectroscopic and thermodynamic properties of OCP photoconversion and reveal general principles underlying the design of photoreceptors by natural evolution. Discussion: Such "molecular surgery" is superior over trial-and-error methods in hypothesis-driven research of complex chemical systems.

Keywords: Orange Carotenoid Protein; atomic mutations; hydrogen bond strength/energy; non-canonical amino acids; orthogonal translation.

Grants and funding

H-WT received funding under the Marie-Skłodowska-Curie grant agreement No. 764591 by the SynCrop ETN consortium of the European Union (Horizon 2020 research and innovation program), NB by the Canada Research Chairs Program grant agreement No. 950-23197, EM and NS by the Ministry of Science and Higher Education of the Russian Federation in the framework of the Agreement no. 075-15-2021-1354, and TF by the German Research Foundation (DFG, grant no. 1276/6-1).