In Situ NMR Studies of Plasma Protein Dynamics and Interactions

点击翻译按钮获取中文摘要

The crowded environment of blood plasma significantly alters the dynamics that control the function of plasma proteins. Hence, the long-term goal of our research program is  to investigate plasma proteins in their native physiological environment and elucidate the in situ molecular mechanisms underlying how protein-plasma interactions modulate functional dynamics and allostery. Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and complementary techniques (e.g. fluorescence and electron microscopy), my group has made significant contributions to the current understanding of how plasma Aß, an aggregation-prone intrinsically disordered polypeptide (IDP), interacts with plasma chaperones, such as human serum albumin (HSA) and epigallocatechin gallate. However, our studies have so far been limited to buffered solutions that do not recapitulate the full complexity of physiological plasma conditions. Preliminary data suggest the hypothesis that the plasma environment controls dynamic transitions from extended to compact conformations through the combined action of steric exclusion and weak binding to protein solutes. As a result, plasma is expected to alter the unfolding thermodynamics of globular proteins and the conformational ensembles accessible to IDPs, which dictate the allosteric response to mutations and post-translational modifications. Nonetheless, only scant knowledge is currently available on how plasma proteins interact with the rest of the plasma environment and on how these interactions affect allostery and unfolding. Hence, our first short-term objective is: (i) to map the interactions and allosteric networks of a-synuclein in plasma and in crowded solutions. a-synuclein is a representative IDP released into plasma as a mediator of cell-to-cell communication. This will be the first time that the allosteric networks of an IDP are mapped under physiological plasma conditions. To complement aim (i), the second short-term objective will focus on a globular plasma protein, i.e. we plan: (ii) to map the interactions of HSA with the plasma environment and determine how they modulate HSA unfolding. This aim will reveal which HSA sites interact with which plasma crowders and how these interactions modulate HSA stability. Preliminary data indicate that both aims are feasible in terms of available materials and experimental methods, capitalizing on NMR approaches employed in our past publications. In the long term, we will extend our in plasma NMR program to other prototypical extracellular IDPs and globular proteins to decipher general transferable rules for how plasma proteins function under native conditions, as needed to bridge the in vitro vs. in vivo gap and design new blood-based assays and blood fractionation technologies. Hence, we expect a broad impact, which will be both scientific and educational, as the proposed program provides unique training opportunities for our diverse HQP cohort.