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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 (1.8A) 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.
