Maximum Flux Transition Paths of Conformational Change

Given two metastable states A and B of a biomolecular system, the problem is to calculate the likely paths of the transition from A to B. Such a calculation is more informative and more manageable if done for a reduced set of collective variables chosen so that paths cluster in collective variable space. The computational task becomes that of computing the "center" of such a cluster. A good way to define the center employs the concept of a committor, whose value at a point in collective variable space is the probability that a trajectory at that point will reach B before A. The committor "foliates" the transition region into a set of isocommittors. The maximum flux transition path is defined as a path that crosses each isocommittor at a point which (locally) has the highest crossing rate of distinct reactive trajectories. (This path is different from that of the MaxFlux method of Huo and Straub.) It is argued that such a path is nearer to an ideal path than others that have been proposed with the possible exception of the finite-temperature string method path. To make the calculation tractable, three approximations are introduced, yielding a path that is the solution of a nonsingular two-point boundary-value problem. For such a problem, one can construct a simple and robust algorithm. One such algorithm and its performance is discussed.Comment: 7 figure

'Mindless markers of the nation': The routine flagging of nationhood across the visual environment

The visual environment has increasingly been used as a lens with which to understand wider processes of social and economic change with studies employing in-depth qualitative approaches to focus on, for example, gentrification or trans-national networks. This exploratory paper offers an alternative perspective by using a novel method, quantitative photo mapping, to examine the extent to which a particular socio-cultural marker, the nation, is ‘flagged’ across three contrasting sites in Britain. As a multi-national state with an increasingly diverse population, Britain offers a particularly fruitful case study, drawing in debates around devolution, European integration and Commonwealth migration. In contributing to wider debates around banal nationalism, the paper notes the extent to which nations are increasingly articulated through commerce, consumption and market exchange and the overall significance of everyday markers (signs, objects, infrastructure) in naturalising a national view of the world

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

The dominant ethnic moment: towards the abolition of 'whiteness'?

International audienc

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases

Recruitment of rare 3-grams at functional sites: Is this a mechanism for increasing enzyme specificity?

<p>Abstract</p> <p>Background</p> <p>A wealth of unannotated and functionally unknown protein sequences has accumulated in recent years with rapid progresses in sequence genomics, giving rise to ever increasing demands for developing methods to efficiently assess functional sites. Sequence and structure conservations have traditionally been the major criteria adopted in various algorithms to identify functional sites. Here, we focus on the distributions of the 20³different types of <it>3</it>-grams (or triplets of sequentially contiguous amino acid) in the entire space of sequences accumulated to date in the UniProt database, and focus in particular on the rare <it>3</it>-grams distinguished by their high entropy-based information content.</p> <p>Results</p> <p>Comparison of the UniProt distributions with those observed near/at the active sites on a non-redundant dataset of 59 enzyme/ligand complexes shows that the active sites preferentially recruit <it>3</it>-grams distinguished by their low frequency in the UniProt. Three cases, Src kinase, hemoglobin, and tyrosyl-tRNA synthetase, are discussed in details to illustrate the biological significance of the results.</p> <p>Conclusion</p> <p>The results suggest that recruitment of rare <it>3</it>-grams may be an efficient mechanism for increasing specificity at functional sites. Rareness/scarcity emerges as a feature that may assist in identifying key sites for proteins function, providing information complementary to that derived from sequence alignments. In addition it provides us (for the first time) with a means of identifying potentially functional sites from sequence information alone, when sequence conservation properties are not available.</p

Sequence and Structure Signatures of Cancer Mutation Hotspots in Protein Kinases

Protein kinases are the most common protein domains implicated in cancer, where somatically acquired mutations are known to be functionally linked to a variety of cancers. Resequencing studies of protein kinase coding regions have emphasized the importance of sequence and structure determinants of cancer-causing kinase mutations in understanding of the mutation-dependent activation process. We have developed an integrated bioinformatics resource, which consolidated and mapped all currently available information on genetic modifications in protein kinase genes with sequence, structure and functional data. The integration of diverse data types provided a convenient framework for kinome-wide study of sequence-based and structure-based signatures of cancer mutations. The database-driven analysis has revealed a differential enrichment of SNPs categories in functional regions of the kinase domain, demonstrating that a significant number of cancer mutations could fall at structurally equivalent positions (mutational hotspots) within the catalytic core. We have also found that structurally conserved mutational hotspots can be shared by multiple kinase genes and are often enriched by cancer driver mutations with high oncogenic activity. Structural modeling and energetic analysis of the mutational hotspots have suggested a common molecular mechanism of kinase activation by cancer mutations, and have allowed to reconcile the experimental data. According to a proposed mechanism, structural effect of kinase mutations with a high oncogenic potential may manifest in a significant destabilization of the autoinhibited kinase form, which is likely to drive tumorigenesis at some level. Structure-based functional annotation and prediction of cancer mutation effects in protein kinases can facilitate an understanding of the mutation-dependent activation process and inform experimental studies exploring molecular pathology of tumorigenesis

Co-encapsulation of isoniazid and rifampicin in liposomes and characterization of liposomes by derivative spectroscopy

Taking into consideration the benefits of the combined therapy of isoniazid (INH) and rifampicin (RIF), this study focused on co-encapsulation of INH and RIF in the same liposome formulation. INH was incorporated in the aqueous phase and RIF in the lipid layer. Liposomes containing either INH or RIF were also prepared. All liposome formulations were compared for their loading capacity, encapsulation percentage and release properties Drug amounts in the liposomes were estimated using peak-to-peak first-order derivative UV spectroscopy. Among the liposome formulations DPPC:chol liposomes showed the highest loading capacity (106.70 +/- 0.12 for INH and 18.17 +/- 0.06 (x 10(-3)) for RIF) and encapsulation percentage (73.84 +/- 0.78 for INH and 81.53 +/- 2.06 for RIF) compared to EPC:chol liposomes (loading capacity 93.36 +/- 0.58 for INH and 17.87 +/- 0.11 (x 10(-3)) for RIF; encapsulation percentage 64.61 +/- 0.51 for INH and 74.45 +/- 0.48 for RIF)

Membrane active peptides and their biophysical characterization

In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide&ndash;membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide&ndash;lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC