The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence ( ex ؍ 480 nm, em ؍ 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C ␣ and C  atoms in the sp 3 hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation ( ؍ 250 -300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C ␣ ؍C  bond and the conjugated system of -electrons. Structurebased site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.Green fluorescent proteins (GFP) 2 and the GFP-like proteins (FPs) have become in recent years very useful tools in many areas of cell biology, biotechnology, and medicine. These proteins exhibit a wide spectral range of fluorescence, from blue to far-red. It became possible to effectively use FPs as single or coupled biomarkers for multicolor labeling of proteins, subcellular compartments, and specific tissue regions. Utilization of FPs enabled monitoring of a variety of characteristics, such as cellular pH and ion concentration, tracking of expression, intracellular localization, and trafficking of proteins of interest in the cell or whole organism and following their interactions with other cellular components (1-5).Chromoproteins are another large group of non-fluorescent counterparts of FPs that share with them the principal fold but not spectral properties (6, 7). However, a number of artificially created, genetically engineered variants of chromoproteins do exhibit fluorescence (6, 8). Members of both fluorescent and non-fluorescent families possess visible coloration corresponding to an absorption range of 450 -610 nm. Extensive diversity of their photophysical characteristics arises mostly from variations in the chemical structure of the internal chromophore group and in the stereochemistry of its adjacent environment.The ...
A green-emitting fluorescent variant, NowGFP, with a tryptophan-based chromophore (Thr65-Trp66-Gly67) was recently developed from the cyan mCerulean by introducing 18 point mutations. NowGFP is characterized by bright green fluorescence at physiological and higher pH and by weak cyan fluorescence at low pH. Illumination with blue light induces irreversible photoconversion of NowGFP from a green-emitting to a cyan-emitting form. Here, the X-ray structures of intact NowGFP at pH 9.0 and pH 4.8 and of its photoconverted variant, NowGFP_conv, are reported at 1.35, 1.18 and 2.5 Å resolution, respectively. The structure of NowGFP at pH 9.0 suggests the anionic state of Trp66 of the chromophore to be the primary cause of its green fluorescence. At both examined pH values Trp66 predominantly adopted a cis conformation; only $20% of the trans conformation was observed at pH 4.8. It was shown that Lys61, which adopts two distinct pH-dependent conformations, is a key residue playing a central role in chromophore ionization. At high pH the side chain of Lys61 forms two hydrogen bonds, one to the indole N atom of Trp66 and the other to the carboxyl group of the catalytic Glu222, enabling an indirect noncovalent connection between them that in turn promotes Trp66 deprotonation. At low pH, the side chain of Lys61 is directed away from Trp66 and forms a hydrogen bond to Gln207. It has been shown that photoconversion of NowGFP is accompanied by decomposition of Lys61, with a predominant cleavage of its side chain at the C -C bond. Lys61, Glu222, Thr203 and Ser205 form a local hydrogen-bond network connected to the indole ring of the chromophore Trp66; mutation of any of these residues dramatically affects the spectral properties of NowGFP. On the other hand, an Ala150Val replacement in the vicinity of the chromophore indole ring resulted in a new advanced variant with a 2.5-fold improved photostability.
The metabolic networks are the most well-studied biochemical systems, with an abundance of in vitro and in vivo data available for quantitative estimation of its kinetic parameters. In this chapter, we present our approach to developing mathematical description of metabolic pathways. The model-based integration of reaction kinetics and the utilization of different types of experimental data including temporal dependencies have been described in detail. Software package DBSolve7 which allows us to develop kinetic model of the biochemical system and integrate experimental data has been presented.
Effects of the hydrogen bond network on the rate constants of energy migration (km), charge separation (ke), electron transfer to QA (kQ) and P+I- recombination in RC of Rhodobacter sphaeroides were analysed in control and modified RC preparations at different temperatures. Modification of RC were made by the addition of 40% v/v DMSO. The rate constants km, ke, kQ were evaluated from pump-and-probe measurements of the absorption difference kinetics at 665 nm corresponding to BPhL- formation and subsequent electron transfer to QA. For the investigation of P+I- recombination a primary quinone acceptor was pre-reduced in the dark by adding of 1 mg/ml of dithionite and 1 mM sodium ascorbate. Recombination kinetics were measured at 665 and 870 nm. The numerical analysis of the temperature dependence of ke and kQ was performed on the basis of the model proposed by Kakitani and Kakitani (T. Kakitani and H. Kakitani (1981), Biochim. Biophys. Acta, 635, 498-514). It was found that: (a) in control samples the molecular rate constants km, ke and kQ were about (3.4 ps)-1, (4.5 ps)-1 and (200 ps)-1, respectively; (b) under modification by DMSO these rates decrease up to (5.3 ps)-1, (10.3 ps)-1 and (500 ps)-1, respectively; (c) as the temperature drops from 300 K to 77 K the rate constant km decreases by 1.8 times in control and by 3.2 times in modified samples. In contrast to the observed km changes the increase in ke and kQ values by 2 and more times under cooling was found in control and modified RC; (d) in control preparations with QA acceptor pre-reduced in the dark the lowering of the temperature caused the increase in the time of P+I- recombination from 10 to 20 ns. After DMSO modification the kinetics of charge recombination in RC was biexponential at room temperature with tau=10 ns and tau1=0.8 ns, and at 77 K with tau=20 ns and tau1=0.6 ns, correspondingly. The results obtained reveal that in RC isolated from Rb. sphaeroides the processes of energy migration, charge separation, electron transfer to QA and ion-radical pair P+I- recombination depend on the state of hydrogen bonds of water-protein structure. Fast relaxation processes in RC structure including polarization of H-containing molecules in the surrounding of electron carriers can accept electron energy dissipated at the initial steps of energy and electron transfer. Copyright 1998 Elsevier Science B.V. All rights reserved.
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