A single molecule approach for measuring the transport properties and energetics of Membrane proteins in Heterogenous Planar Bio-mimetic assemblies
Poudel, Kumud Raj
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The significance of transmembrane protein research is well documented. Numerous studies have clearly established the biological, biophysical and pharmaceutical importance that these membrane components serve. Communications through receptors regulate countless body functions and they also provide structural support to the cell. However, a lack of high-resolution structure data has limited our understanding of these proteins that make it necessary to study them in in-vitro platforms or `bio-mimetic' assemblies.Albeit that an assortment of platforms have been suggested for in-vitro studies, the issues, however, remain the same. The lack of mobility of the proteins in artificial environments, the question of functionality that arises with mobility and the search in general for the best assembly, is still a work in progress. In this work, we have taken some of the most accepted platforms in the field and characterized them through the lens of single molecule spectroscopy.We have addressed the question of mobility by reducing it down to a single molecule and comparing it with the bulk. By utilizing the Serotonin Receptor 5HT3A¬ we have shown that techniques such as passivation of the substrates in the assemblies by Bovine Serum Albumin has a significant effect at the molecular level. The larger size of the intracellular domain for the 5HT3A¬ served as a great probe to understand and evaluate the interaction of a surface passivator with the integrated membrane protein. We have also taken this a step further by developing a novel, single cushion 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) assembly and added another degree of complexity- through a phase transition. We have utilized phase transition to get an insight into the local protein environment, activation energies, heterogeneity and diffusion characteristics by using Annexin V as our probe.The work presented here studies two completely different biological platforms using two entirely different transmembrane proteins. This deliberate contrast was created in order to provide a complete outlook into the transport properties and energetics of these crucial biological components in planar, heterogeneous bio-mimetic assemblies.