Ratcheted molecular-dynamics simulations identify efficiently the transition state of protein folding

The atomistic characterization of the transition state is a fundamental step to improve the understanding of the folding mechanism and the function of proteins. From a computational point of view, the identification of the conformations that build out the transition state is particularly cumbersome, mainly because of the large computational cost of generating a statistically-sound set of folding trajectories. Here we show that a biasing algorithm, based on the physics of the ratchet-and-pawl, can be used to identify efficiently the transition state. The basic idea is that the algorithmic ratchet exerts a force on the protein when it is climbing the free-energy barrier, while it is inactive when it is descending. The transition state can be identified as the point of the trajectory where the ratchet changes regime. Besides discussing this strategy in general terms, we test it within a protein model whose transition state can be studied independently by plain molecular dynamics simulations. Finally, we show its power in explicit-solvent simulations, obtaining and characterizing a set of transition--state conformations for ACBP and CI2

The influence of Atlantic High on seasonal rainfall in Argentina

The objective of this work is to quantify the influence of the position and intensity of the Atlantic High (AH) on seasonal rainfall in Argentina. Monthly precipitation data in 68 stations from the National Meteorological Service of Argentina, the Secretariat of Water Resource and the Territorial Authority of the Limay, Neuquén, and Negro rivers basins (AIC) were used. Correlation was the methodology used to identify the link between seasonal rainfall and some indices especially defined to detect the position and intensity of the anticyclone. Precipitation composites of the years when the value of these indices was extremely high (greater than their second tercile) and extremely low (lower than their first tercile) were built for every season. They were analyzed and compared with humidity anomaly transport composites for the same sets of years in order to study circulation patterns associated with seasonal rainfall anomalies. Results indicate that when the AH is intensified, winter rainfall increases in central Argentina and spring rainfall increases in northern Cuyo region. When the AH is displaced towards the north of its mean position, autumn rainfall is favored in the northeast and central and south Patagonia, meanwhile winter and spring rainfall decreases in central Argentina. When the AH is displaced towards the east of its mean position, summer rainfall decreases in Central Andes. Finally, AH indices were used to detect the influence on rainfall in advance. Correlations between seasonal rainfall and the defined indices that characterize the anticyclone in the previous month suggest that there is some rainfall predictability, especially during spring in almost every region in Argentina.Fil: Garbarini, Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones del Mar y la Atmósfera. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones del Mar y la Atmósfera; ArgentinaFil: González, Marcela Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones del Mar y la Atmósfera. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones del Mar y la Atmósfera; ArgentinaFil: Rolla, Alfredo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones del Mar y la Atmósfera. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones del Mar y la Atmósfera; Argentin

Metadynamic sampling of the free energy landscapes of proteins coupled with a Monte Carlo algorithm

Metadynamics is a powerful computational tool to obtain the free energy landscape of complex systems. The Monte Carlo algorithm has proven useful to calculate thermodynamic quantities associated with simplified models of proteins, and thus to gain an ever-increasing understanding on the general principles underlying the mechanism of protein folding. We show that it is possible to couple metadynamics and Monte Carlo algorithms to obtain the free energy of model proteins in a way which is computationally very economical.Comment: Submitted to Gen

Determination of Protein Structural Ensembles by Hybrid-Resolution SAXS Restrained Molecular Dynamics

Small-angle X-ray scattering (SAXS) experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated into molecular dynamics (MD) simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of metainference with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin, showing that the inclusion of SAXS restraints is effective in generating a reliable conformational ensemble, improving the agreement with independent experimental data

What is a Gene? A Two Sided View

The need to account for all currently available experimental observations concerning the gene nature, has reshaped the concept of gene turning it from the essentially mechanistic unit, predominant during the '70s, into a quite abstract open and generalized entity, whose contour appears less defined as compared to the past. Here we propose the essence of the gene to be considered double faced. In this respect genotypic and phenotypic entities of a gene would coexist and mix reciprocally. This harmonizes present knowledge with current definitions and predisposes for remodelling of our thinking as a consequence of future discoveries. A two sided view of the gene also allows to combine the genetic and epigenetic aspects in a unique solution, being structural and functional at the same time and simultaneously able to include the different levels in an overlapping unicum

Expression of Werner and Bloom syndrome genes is differentially regulated by in vitro HIV-1 infection of peripheral blood mononuclear cells

In HIV infection, continuous immune activation leads to accelerated ageing of the adaptive immune system, similar to that observed in elderly people. We investigated the expression of WRN and BLM (genes involved in disorders characterized by premature ageing, genomic instability and cancer predisposition) in peripheral blood mononuclear cells (PBMC) activated in vitro with phytohaemagglutinin (PHA) and infected with different HIV-1 strains. The steady state levels of mRNA were analysed by reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was assayed using immunocytochemistry and Western blot techniques. In uninfected PBMC, PHA stimulation induced an increase in BLM mRNA and protein expression, while WRN expression remained virtually unchanged. When PBMC were infected in vitro with a lymphotropic HIV-1 strain, the level of BLM mRNA showed a peak at 24 h of infection, followed by a decline to uninfected culture levels. A similar result failed to be seen using an R5-tropic HIV-1 strain. In accordance with mRNA expression, in HIV-infected cultures PBMC were stained more frequently and more intensely by a BLM-specific antibody as compared to uninfected cultures, staining peaking at 24. Conversely, WRN expression was not modulated by HIV-1. The proportion of cells showing BLM up-regulation, established by immunocytochemical staining, was much greater than the proportion of productively infected PBMC, as established by proviral DNA measurement. This result indicates that BLM up-regulation is probably a result of an indirect bystander cell effect. Activation of the BLM gene in infected PBMC suggests that premature ageing could be a further immunopathogenetic mechanism involved in HIV-induced immunodeficiency, and points to a possible new candidate target for innovative therapeutic intervention

Sequence Specificity in the Entropy-Driven Binding of a Small Molecule and a Disordered Peptide

Approximately one-third of the human proteome is made up of proteins that are entirely disordered or that contain extended disordered regions. Although these disordered proteins are closely linked with many major diseases, their binding mechanisms with small molecules remain poorly understood, and a major concern is whether their specificity can be sufficient for drug development. Here, by studying the interaction of a small molecule and a disordered peptide from the oncogene protein c-Myc, we describe a "specific-diffuse" binding mechanism that exhibits sequence specificity despite being of entropic nature. By combining NMR spectroscopy, biophysical measurements, statistical inference, and molecular simulations, we provide a quantitative measure of such sequence specificity and compare it to the case of the interaction of urea, which is diffuse but not specific. To investigate whether this type of binding can generally modify intermolecular interactions, we show that it leads to an inhibition of the aggregation of the peptide. These results suggest that the binding mechanism that we report may create novel opportunities to discover drugs that target disordered proteins in their monomeric states in a specific manner.G.T.H. is supported by the Churchill Scholarship and the Gates Cambridge Trust Scholarship

Induction of Cross-Reactive Antibodies to 2009 Pandemic H1N1 Influenza Virus (pH1N1) After Seasonal Vaccination (Winters 2003/04 and 2007/08)

Abstract We measured haemagglutination inhibiting (HI) serum antibody titers to vaccine matched A/H1N1 influenza virus strain and to the new pandemic 2009 A/H1N1 (pH1N1) virus in two groups of volunteers prior and after 2003/2004 or 2007/2008 influenza seasonal vaccine administration. The responses were examined considering the overall volunteers studied in the two winters (144 and 79, respectively) and grouping those subjects in birth cohort classes (1903–1919; 1920–1957; 1958–1977). Before vaccination, HI antibody titers were found in all the groups examined and, on comparing the different age-groups, titers were higher in the younger groups as compared with the oldest against the A/H1N1 seasonal strains but titers were higher in the oldest as compared with the younger ones against the pH1N1 strain. Vaccination induced significant increases in HI titers against the matched A/H1N1 vaccine strains in all the groups examined. The responses satisfied the EMEA criteria and were higher in the youngest volunteers as compared with older groups. Increases were also found in the level of cross-reactive HI antibodies to the new pandemic 2009 A/H1N1 virus although in most instances the requirements of the EMEA were not met

The Folding Mechanism of the SH3 Domain from Grb2

SH3 domains are small protein modules involved in the regulation of important cellular pathways. These domains mediate protein-protein interactions recognizing motifs rich in proline on the target protein. The SH3 domain from Grb2 (Grb2-SH3) presents the typical structure of an SH3 domain composed of two-three stranded antiparallel \u3b2-sheets orthogonally packed onto each other, to form a single hydrophobic core. Grb2 interacts, via SH3 domain, with Gab2, a scaffolding disordered protein, triggering some key metabolic pathways involved in cell death and differentiation. In this work we report a mutational analysis (\u3c6-value analysis) of the folding pathway of Grb2-SH3 that, coupled with molecular dynamic simulations, allows us to assess the structure of the transition state and the mechanism of folding of this domain. Data suggest that Grb2-SH3 folds via a native-like, diffused transition state with a concurrent formation of native-like secondary and tertiary structure (nucleation-condensation mechanism) and without the accumulation of folding intermediates. The comparison between our data and previous folding studies on SH3 domains belonging to other proteins, highlights that proteins of this class may fold via alternative pathways, stabilized by different nuclei leading or not to accumulation of folding intermediates. This comparative analysis suggests that the alternative folding pathways for this class of SH3 domains can be selectively regulated by the specific aminoacid sequences