The ultimate goal is to understand the fundamental principles underlying the sequence-structure determinism, a major unsolved issue in contemporary biology. The immediate objective is to approach this question by protein design and produce a voltage-gated channel from its constituents voltage sensor (VSM) and pore (PM) modules, to retrieve its function after reconstitution in lipid bilayers, and to determine its three-dimensional structure in membranes by a combination of solution and solid-state NMR spectroscopy. The choice of voltage-gated channels is based on their importance as key control elements of cellular excitability, the mechanism underlying voltage-sensing is not fully understood, and a structure at atomic resolution is not available. A structure for the PM of S. lividans KcsA and of M. thermoautotrophicum MthK is available. The structure of the VSM remains elusive, yet this is the unique element that endows voltage-gated channels with the ability to couple a transmembrane voltage to channel opening. This is what needs to be done and what the proposed work intends to achieve.
The specific aims for the revised application are focused on providing structures of the VSM and the full channel containing both VSM and PM modules of prokaryotic and designed channel proteins. Function is established by reconstitution of purified proteins in lipid bilayers and by expression of gene products in mammalian cells. Protein structure is determined by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated lipid micelles and by solid-state NMR in oriented phospholipid bilayers. The ultimate test of a successful design is recapitulation of biological function of the whole protein by assembling it from VSM and PM and determining its structure. These advances may contribute valuable insights to understand mechanisms of disease and provide structural blueprints for drug design.
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