A large, ubiquitous, and homologous group of ion channels perform crucial functions in eukaryotes including neural signaling, cardiac rhythm regulation, and modulators of GPCR signal transduction pathways. In particular, K+ channels are endemic to all human cells where they are involved in controlling the electrochemical potential by passing ions through the cell membrane. Voltage-gated K+ (Kv) channels generate electric impulses essential to the function of muscles, nerves, and the endocrine system and are fundamental to such behaviors as cell proliferation, apoptosis, and cancer cell proliferation. These systems have proven exceedingly challenging to study by traditional structure methods. The goal of the proposed research is to apply and extend solid-state NMR methodology to obtain structural and functional information on voltage-gated K+ channels. This work will begin with the channel KcsA from Streptomyces lividans. The ease of preparation, sample stability, homology with other K+ channels, and extant studies, including SSNMR, makes KcsA an ideal system for probing the basic biophysics involved in ion selection, binding transmission, and release. Experiments planned for KcsA include chemical shift assignments and tensor measurements over a range of pH, K+ concentration, and in the presence of quaternary ammonium ions. This data will be leveraged to develop structure determination methods for highly ambiguous SSNMR data. In this scheme, referred to in this proposal as """"""""top-down"""""""" SSNMR, structural models will be screened against unassigned NMR data for consistency. NMR constrained molecular dynamics (MD) will then be performed with a combination of site-specific and ambiguous information. Following this, more elaborate prokaryotic systems such as KvAP and mammalian systems such as Kv1.2 or possibly Kv10.1 will be targeted. Structural studies of channels such as Kv1.2 illustrate and suggest a mechanism for the function of voltage sensors, pore gating, and regulation by additional subunits. The open states of these systems have been stabilized in a more authentically functional form. If funded, this project would provide me with crucial training in structural and mechanistic biology. The impact of this proposed work on public health is through my long-term goal of applying SSNMR to crucial cancer and heart disease related targets. This program would ultimately harness the power of SSNMR to study large membrane proteins, a sizeable and critically important players in human disease.
|Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane et al. (2015) Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces. J Biomol NMR 61:361-7|