We will characterize the structure and dynamics of two intrinsic membrane proteins in their native bilayer environments, under conditions consistent with their functions: KcsA, the prototypical K+ channel of S. lividans, and the c subunit of ATP synthase from E. coli. Solid State NMR will provide atomic level details on structure and dynamics, without any requirement for crystals or mono-dispersed solutions. KcsA is a homology model for medically relevant K+ channels of mammals, and is the best characterized system for clarifying the highly efficient and selective ion transmission, and the principles underlying channel gating. Structural work by X-ray crystallography on the closed state of the channel stands among the best accomplishments of membrane protein structural biology, and yet is limited because a truncated protein was studied under nonfunctional conditions, providing little or no information on dynamical flexibility. The bilayer environment and the composition of lipids are known to be crucial for structure, function, and dynamics of intrinsic membrane proteins, including the function and folding of KcsA. We propose to study the full length, active form of the protein in a bilayer environment, contrasting it to the protein in the crystal, using a number of recently developed approaches to stabilize the open state in the bilayer. We will clarify structural differences between the high and low pH states, the open and closed states, and between the high and low K+ states, and the dynamic interconversion between these states in the bilayer, and their interactions with lipids. In ATP synthase, the c subunit plays the central role in proton transfer across the bilayer, and it is believed that conformation changes in this subunit drive the conformation changes of F1, enabling ATP synthesis. Protonation of residue D61 is believed to drive overall rotation of the oligomer, as well as a conformation change in the c subunit, involving an interhelix loop that interacts directly with F1. Solution NMR studies have shown that the c subunit monomer in organic solvents is a helical hairpin whose interhelical loop structure is a function of pH. To date, there is no high-resolution study of the c subunit assembly in the bilayer nor in FO. We will assign spectra of this oligomeric assembly (c10 and FO) above and below the pKa of the crucial pump residue, D61. We will study quaternary contacts between subunit c and neighboring subunits. For both systems, we will apply recently developed NMR methods for determining structure, including selective recoupling techniques for determining distances, dipolar tensor-based vector angle correlation methods for constraining torsion angles, and chemical shift analysis. Preliminary data include partial sequence-specific assignments for both systems in bilayers, and evidence for NMR for pH-dependent conformations.

Public Health Relevance

Membrane proteins are foremost among crucially important medical targets, and yet the structures and mechanisms of most remain poorly characterized by traditional methods. We plan to apply a solid state NMR to elucidate two important cases: (1) KcsA, a prototypical K+ channel, and an important homology model for the medically relevant K+ channels of mammals, and (2) ATP synthase subunit c, a proton pump that drives a rotary mechanism for the synthesis of ATP and has been pursued as an organism specific target for inhibition in connection with tuberculosis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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Columbia University (N.Y.)
Other Domestic Higher Education
New York
United States
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Wylie, Benjamin J; Bhate, Manasi P; McDermott, Ann E (2014) Transmembrane allosteric coupling of the gates in a potassium channel. Proc Natl Acad Sci U S A 111:185-90
Bhate, Manasi P; Wylie, Benjamin J; Thompson, Ameer et al. (2013) Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay. Protein Expr Purif 91:119-24
Huang, Kuo-Ying; Siemer, Ansgar B; McDermott, Ann E (2011) Homonuclear mixing sequences for perdeuterated proteins. J Magn Reson 208:122-7
Bauer, Andras J; Gieschler, Simone; Lemberg, Kathryn M et al. (2011) Functional model of metabolite gating by human voltage-dependent anion channel 2. Biochemistry 50:3408-10
Bhate, Manasi P; Wylie, Benjamin J; Tian, Lin et al. (2010) Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration. J Mol Biol 401:155-66