KiNEEt: Aplicación para el aprendizaje y rehabilitación en educación especial

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Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange

Presynaptic A2A adenosine receptors dampen CB1 cannabinoid receptor-mediated inhibition of corticostriatal glutamatergic transmission

Background and Purpose Both CB1 cannabinoid and A2A adenosine receptors (CB1Rs and A2ARs) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A post-synaptic CB1R-A2AR interaction has already been unraveled, but the presynaptic A2AR-mediated control of presynaptic neuromodulation by CB1Rs remains to be defined. Since the corticostriatal terminals provide the major input of the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. Experimental Approach Here we employ selective presynaptic tools to study the putative presynaptic interaction between the two neuromodulator systems. Pharmacological manipulation of CB1R and A2AR was carried out in isolated nerve terminals used for flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement, as well as in whole-cell patch-clamp recordings in horizontal corticostriatal slices. Results Flow synaptometry showed that A2AR are extensively co-localized with CB1R-immunopositive corticostriatal terminals, and A2AR co-immunoprecipitated CB1R in these purified terminals. A2AR activation decreased CB1R radioligand binding and decreased the CB1R-mediated inhibition of high-K+-evoked glutamate release in corticostriatal terminals. Accordingly, A2AR activation prevented CB1R-mediated paired-pulse facilitation and attenuated the CB1R-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. Conclusions and Implications These results show that presynaptic A2AR dampens CB1R-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to shut-down the potent CB1R-mediated presynaptic inhibition, enabling a frequency-dependent enhancement of synaptic efficacy at corticostriatal synapses

The Location and Nature of General Anesthetic Binding Sites on the Active Conformation of Firefly Luciferase; A Time Resolved Photolabeling Study

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies

Nucleotide Binding Switches the Information Flow in Ras GTPases

The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (SGDP and SGTP, respectively). For all the considered systems, the intrinsic flexibility of SGDP was higher than that of SGTP, suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, SGDP of Gαt, is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP

Mechanochemical Coupling in the Myosin Motor Domain. II. Analysis of Critical Residues

An important challenge in the analysis of mechanochemical coupling in molecular motors is to identify residues that dictate the tight coupling between the chemical site and distant structural rearrangements. In this work, a systematic attempt is made to tackle this issue for the conventional myosin. By judiciously combining a range of computational techniques with different approximations and strength, which include targeted molecular dynamics, normal mode analysis, and statistical coupling analysis, we are able to identify a set of important residues and propose their relevant function during the recovery stroke of myosin. These analyses also allowed us to make connections with previous experimental and computational studies in a critical manner. The behavior of the widely used reporter residue, Trp501, in the simulations confirms the concern that its fluorescence does not simply reflect the relay loop conformation or active-site open/close but depends subtly on its microenvironment. The findings in the targeted molecular dynamics and a previous minimum energy path analysis of the recovery stroke have been compared and analyzed, which emphasized the difference and complementarity of the two approaches. In conjunction with our previous studies, the current set of investigations suggest that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely the general feature for mechanochemical coupling in many molecular motors. The fundamental strategies of examining both collective and local changes and combining physically motivated methods and informatics-driven techniques are expected to be valuable to the study of other molecular motors and allosteric systems in general

A Dual Color Far-Red to Near-Infrared Firefly Luciferin Analogue Designed for Multi-Parametric Bioluminescence Imaging

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase. However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual color, far-red to near infrared (nIR) emitting analogue of beetle luciferin, which akin to natural luciferin exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima with different Fluc mutants up to .max 706 nm. This is the furthest red-shifted form of bioluminescence reported without the requirement of a resonance energy transfer acceptor and such improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging