Helix/Coil Nucleation: A Local Response to Global Demands

A complete description of protein structure and function must include a proper treatment of mechanisms that lead to cooperativity. The helix/coil transition serves as a simple example of a cooperative folding process, commonly described by a nucleation-propagation mechanism. The prevalent view is that coil structure must first form a short segment of helix in a localized region despite paying a free energy cost (nucleation). Afterward, helical structure propagates outward from the nucleation site. Both processes entail enthalpy-entropy compensation that derives from the loss in conformational entropy on helix formation with concomitant gain in favorable interactions. Nucleation-propagation models inherently assume that cooperativity arises from a sequential series of local events. An alternative distance constraint model asserts there is a direct link between available degrees of freedom and cooperativity through the nonadditivity in conformational entropy. That is, helix nucleation is a concerted manifestation of rigidity propagating through atomic structure. The link between network rigidity and nonadditivity of conformational entropy is shown in this study by solving the distance constraint model using a simple global constraint counting approximation. Cooperativity arises from competition between excess and deficiency in available degrees of freedom in the coil and helix states respectively

Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses

IntroductionMetabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.ObjectiveWe aimed to combine LC–MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.MethodsWe developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity.ResultsAmong the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel.ConclusionOur results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC–MS

Membrane protein structure, function, and dynamics: a perspective from experiments and theory

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.JTD, IA, and MR used the computational resources of the Modeling Facility of the Department of Chemistry, University of California Irvine funded by NSF Grant CHE-0840513 for this work. A-NB was supported in part by the Marie Curie International Reintegration Award IRG-26920.TWA was supported by ARC DP120103548, NSF MCB1052477, DE Shaw Anton (PSCA00061P; NRBSC, through NIH RC2GM093307), VLSCI (VR0200), and NCI (dd7). BA and SV acknowledge the support by ERC advanced Grant No. 268888. ZC and PG would like to acknowledge Reference Framework (NSRF) 2011–2013, National Action ‘‘Cooperation,’’ under grant entitled ‘‘Magnetic Nanoparticles for targeted MRI therapy (NANOTHER),’’ with code ‘‘11RYM-1-1799.’’ The program is cofunded by the European Regional Development Fund and national resources. Part of the calculations presented herein were performed using resources of the LinkSCEEM-2 project, funded by the EC under FP7 through Capacities Research Infrastructure, INFRA-2010-1.2.3 Virtual Research Communities, Combination of Collaborative Project and Coordination and Support Actions (CPCSA) under Grant agreement no. RI-261600. GB was supported in part by NSF grant MCB1330728 from the National Science Foundation and Grant PO1GM55876-14A1 from the National Institutes of Health. LD received funding from EU FP7 (PIOF-GA-2012-329534). LD, and MLK used the computational resources of Temple University, supported by the National Science Foundation through major research instrumentation grant number CNS-09-58854. JS acknowledges support from the Instituto de Salud Carlos III FEDER (CP12/03139