CHARACTERIZATION OF MEMBRANE PROTEIN BY SSNMR

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Description: (Verbatim from the applicant's abstract) We propose to carry out structural studies of the proton pump, F0 from ATP synthase, involving high field solid state NMR distance measurements of the uniformly 13C, 15N labeled c subunit in this complex. Many of the methods have been validated using hydrated, micro-crystalline proteins such as BPTI and ubiquitin. The mechanistically crucial c subunit of this complex will be an ideal trial case for assignments in membrane proteins, since it has only 79 residues, and probably has many well dispersed NMR lines. Magic angle spinning, high-resolution measurements of isotropic carbon chemical shifts, analogous to solution NMR, will be used to confirm sidechain identity and secondary structure. 3D pulse sequences based on efficient C-C and C-N transfers will be used to establish site-specific backbone and sidechain assignments for subunit c. We recently demonstrated that high-resolution multidimensional solid state NMR methods can be used to correlate backbone and side-chain chemical shifts of hydrated micro-crystalline U-13C, 15N BPTI and of the intrinsic membrane protein, LH1. Typical 13C peak linewidths are 0.5 ppm; agreement between shifts measured for BPTI in the solid state and solution is typically better than 0.5 ppm. Both N-C and C-C transfers can be performed with high efficiency, thus allowing sequential assignments through the backbone. Distances will be probed with multidimensional studies of the assigned sidechain carbons; they will also be probed with selective fluorination and 19F-19F through space distance measurements. Uniformly labeled subunit c and synthase complex are available through collaboration with Professor Mark Girvin (Einstein College of Medicine). Some of the labeled samples have already been prepared at many mg scale, and instrument time on high field solids machines will be available in one year. Subunit c will be studied in the context of an intact F0 complex; although this complex is quite large (ca. 200 kDa) we can still expect very strong signals associated with the c subunit since there are 12 copies in the complex as compared with only 1-2 copies of all other peptides, and subunit c represents about half of the mass of F0. Subunit c will also be studied in the context of reconstituted lipid embedded c12 oligomers, so that mixed isotopic labeling strategies can be used to distinguish intramolecular and intermolecular contacts. We will test the relevance of existing structural models for the oligomer, particularly the hypothesis that ionization of Asp61, the central player in proton pumping, provokes mechanistically important global conformational changes in ATP synthase.