Unusual shift in the visible absorption spectrum of an active ctenophore photoprotein elucidated by time‑dependent density functional theory

Active hydromedusan and ctenophore Ca2+-regulated photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxycoelenterazine (an oxygenated intermediate of coelenterazine). Whereas the absorption maximum of hydromedusan photoproteins is at 460–470 nm, ctenophore photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein–substrate complex was optimized using a linear scaling quantum–mechanical method. Electronic excitations pertinent to the spectra of the 2-hydroperoxy adduct of coelenterazine were simulated with time-dependent density functional theory. The dihedral angle of 60° of the 6-(p-hydroxy)-phenyl group relative to the imidazopyrazinone core of 2-hydroperoxycoelenterazine molecule was found to be the key factor determining the absorption of ctenophore photoproteins at 437 nm. The residues relevant to binding of the substrate and its adopting the particular rotation were also identified

A Test System for Tick-Borne Encephalitis Virus Detection Based on Bioluminescent Immunoassay

Вирус клещевого энцефалита (ВКЭ) является инфекционным агентом тяжелого нейрозаболевания – клещевого энцефалита. Он переносится иксодовыми клещами, инфекция распространена по всей лесной и лесостепной зоне умеренного пояса Евразийского континента, в том числе в Сибирском регионе Российской Федерации. Несмотря на наличие коммерческих аналитических систем для выявления ВКЭ, задача разработки быстрых и достоверных методов анализа, пригодных для рутинного применения, в том числе и при экологических исследованиях, остается актуальной. В результате проведенных исследований разработана и испытана тест-система для выявления вируса клещевого энцефалита (ВКЭ) в клещах твердофазным биолюминесцентным иммуноанализом. В качестве сенсора использовали гибридный белок, состоящий из одноцепочечного мини-антитела мыши к вирионному белку Е и термостабильного варианта люциферазы Renilla muelleri. Этот уникальный белок был получен и исследован авторами ранее. В работе рассмотрены варианты экспрессии гибрида рекомбинантными клетками E. coli разных штаммов и найдены оптимальные условия получения высокоочищенного препарата этого белка. Запуск биолюминесцентной реакции люциферазного домена осуществляли с помощью стабильной природной формы субстрата – Са2+-зависимого целентеразин-связывающего белка, рекомбинантый вариант которого был получен авторами ранее. Найдены условия долгосрочного хранения ключевых элементов системы – гибридного белка, стабильной формы субстрата люциферазы, активированных планшетов. Анализ осуществляется в высокопроизводительном микропланшетном варианте. Разработанным способом исследовано более 900 образцов природных клещей. По чувствительности (89,5 %) и специфичности (98,9 %) биолюминесцентный анализ не уступает колориметрическому, будучи при этом проще и быстрееThe tick-borne encephalitis virus (TBEV) is the causative agent of one of the most severe human neuroinfections. The infection transmitted by ixodid ticks is spread throughout the forest and foreststeppe zones of the temperate climatic belt of the Eurasian continent, including the Siberian region of the Russian Federation. Despite the availability of commercial analytical systems for the detection of TBEV, the task of developing approaches to a quick and reliable analysis that can be performed routinely, particularly in environmental studies, remains topical. A solid-phase bioluminescent immunoassay for determining the tick-borne encephalitis virus (TBEV) in ticks was developed. The assay is based on the hybrid protein consisting of a modified thermostable version of Renilla muelleri luciferase and a single-chain mini-antibody to protein E. This unique protein had been obtained and investigated by the authors earlier. The current study describes the expression of the hybrid protein in two different strains of recombinant E. coli cells. The optimal conditions for obtaining a highly purified protein were found. The bioluminescent reaction of the luciferase domain was triggered with the help of the stable natural form of the substrate, a Ca2+-dependent coelenterazine-binding protein, the recombinant variant of which was obtained by the authors. The conditions for production and storage of the immunoassay components (the hybrid protein, the stable form of the luciferase substrate, and activated microplates) were determined. Using the developed test system, more than 900 tick samples were analyzed for TBEV. In terms of sensitivity (89.5%) and specificity (98.9%), the proposed method is not inferior to colorimetric detection and is much simpler and faster than the latte

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