36 resultados para Atomistic molecular dynamics simulations


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Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the alpha1B-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the alpha1B-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the alpha1B-AR thus providing new insights into the structure/function relationship of G protein-coupled receptors.

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Knowledge of T(1) relaxation times can be important for accurate relative and absolute quantification of brain metabolites, for sensitivity optimizations, for characterizing molecular dynamics, and for studying changes induced by various pathological conditions. (1)H T(1) relaxation times of a series of brain metabolites, including J-coupled ones, were determined using a progressive saturation (PS) technique that was validated with an adiabatic inversion-recovery (IR) method. The (1)H T(1) relaxation times of 16 functional groups of the neurochemical profile were measured at 14.1T and 9.4T. Overall, the T(1) relaxation times found at 14.1T were, within the experimental error, identical to those at 9.4T. The T(1)s of some coupled spin resonances of the neurochemical profile were measured for the first time (e.g., those of gamma-aminobutyrate [GABA], aspartate [Asp], alanine [Ala], phosphoethanolamine [PE], glutathione [GSH], N-acetylaspartylglutamate [NAAG], and glutamine [Gln]). Our results suggest that T(1) does not increase substantially beyond 9.4T. Furthermore, the similarity of T(1) among the metabolites (approximately 1.5 s) suggests that T(1) relaxation time corrections for metabolite quantification are likely to be similar when using rapid pulsing conditions. We therefore conclude that the putative T(1) increase of metabolites has a minimal impact on sensitivity when increasing B(0) beyond 9.4T.

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Using Brownian dynamics simulations, we investigate here one of possible roles of supercoiling within topological domains constituting interphase chromosomes of higher eukaryotes. We analysed how supercoiling affects the interaction between enhancers and promoters that are located in the same or in neighbouring topological domains. We show here that enhancer-promoter affinity and supercoiling act synergistically in increasing the fraction of time during which enhancer and promoter stay in contact. This stabilizing effect of supercoiling only acts on enhancers and promoters located in the same topological domain. We propose that the primary role of recently observed supercoiling of topological domains in interphase chromosomes of higher eukaryotes is to assure that enhancers contact almost exclusively their cognate promoters located in the same topological domain and avoid contacts with very similar promoters but located in neighbouring topological domains.

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Understanding the structure of interphase chromosomes is essential to elucidate regulatory mechanisms of gene expression. During recent years, high-throughput DNA sequencing expanded the power of chromosome conformation capture (3C) methods that provide information about reciprocal spatial proximity of chromosomal loci. Since 2012, it is known that entire chromatin in interphase chromosomes is organized into regions with strongly increased frequency of internal contacts. These regions, with the average size of ∼1 Mb, were named topological domains. More recent studies demonstrated presence of unconstrained supercoiling in interphase chromosomes. Using Brownian dynamics simulations, we show here that by including supercoiling into models of topological domains one can reproduce and thus provide possible explanations of several experimentally observed characteristics of interphase chromosomes, such as their complex contact maps.

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In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

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Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D = ∼1 - 5 μM. Beyond the affinity threshold at K D < 1 μM we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.