The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the "catalytic function" by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins.

Protein Design Strategies for the Structural-Functional Studies of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are an important family of membrane proteins responsible for many physiological functions in human body. High resolution GPCR structures are required to understand their molecular mechanisms and perform rational drug design, as GPCRs play a crucial role in a variety of diseases. That is difficult to obtain for the wild-type proteins because of their low stability. In this review, we discuss how this problem can be solved by using protein design strategies developed to obtain homogeneous stabilized GPCR samples for crystallization and cryoelectron microscopy.

Crystal structure of semi-synthetic obelin-v after calcium induced bioluminescence implies coelenteramine as the main reaction product.

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.