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

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.

Microbial canthaxanthin: an orange-red keto carotenoid with potential pharmaceutical applications

“…Carotenoids, from a structural perspective, exhibit a geometric pattern where two residual parts are connected by a double bond, either in the same direction (forming the E form) or in the opposite direction (forming the Z form) with respect to the plane (Moldenhauer et al, 2023). Isomers, aside from having differences in melting points, stability, and solubility, also display significant differences in absorption affinity, color, and color intensity (Venugopal, 2008).…”

Orthogonal translation with 5‐cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding

“…Recently, we used Mm PylRS‐based and Methanocaldococcus jannaschii tyrosyl‐tRNA synthetase ( Mj TyrRS)‐based o‐pairs to incorporate Trp‐like structures such as naphthyl‐alanines and β‐(1‐azulenyl)‐L‐alanine (AzAla) (Baumann et al, 2019a). We and others have also demonstrated the utility of Bta by studying the role of single hydrogen bonds in protein photophysics (Moldenhauer et al, 2023), while AzAla has proven to be an excellent molecular tool for studying the vibrational energy transfer in proteins (Deniz et al, 2021). More recently, the chimeric design of pyrrolysyl‐tRNA synthetase/tRNA pairs with phenylalanine‐tRNA identity elements enabled the ribosomal incorporation of tryptophan analogs such as 6‐cyanotryptophan and 7‐cyanotryptophan using E. coli and mammalian cells (Ding et al, 2020).…”