This Synthetic Core is essential to the success of the proposed Center because the materials to be made by this Core are either not available commercially or prohibitively expensive. As the Center establishes their utility in successful structure determination of MPs, the IOC of Core A will organize every effort to convince the vendors to commercialize these products. Chou will direct this Core to serve the following roles in the Center. 1. Synthesize detergents. The use of deuterated detergent was a key to measuring NOE restraints in the structure determination of PLN, M2, and VDAC (Hiller et al., 2008;Oxenoid and Chou, 2005;Schnell and Chou, 2008) for two reasons. (1) For NOESY experiments with long NOE mixing, the protons of regular detergent can substantially dilute protein NOEs by spin diffusion. (2) Most detergents have strong resonances very close to those of protein methyl groups. Therefore, under typical detergent concentration used in NMR Program Director/Principal Investigator (Last, First, Middle): Chou, James J. ? Cores PHS 398/2590 (Rev. 11/07) Page Continuation 134 Format Page samples (>100 mM), the resonances from the natural abundance 13C are much stronger than those of protein, which cause problems such as resonance overlap and distorted baseline in NMR spectra. Dr. Vlado Gelev, a well-trained organic chemist who synthesized deuterated LDAO for the VDAC project, will produce deuterated versions of the most commonly used detergents and lipids in NMR studies of MPs (see Table B.1 in Appendix C for a list of detergents to be synthesized). He will also work with Sanders in Project 3 in synthesizing the newly proposed detergents to be tested for MP reconstitution. In addition to detergents, Gelev will help Sanders to synthesize a series of stable nitroxyl radical spin labels and lanthanide-chelating agents for measuring PRE restraints and for weakly aligning MPs for RDC measurement, respectively (rationale described in Project 3 3.3 and 3.5.3). Details of the synthetic schemes are presented in Appendix C. 2. Synthesize precursors. Alternative labeling strategies of amino acid residues for in vitro translation/transcription and in vivo protein production will offer new opportunities in protein NMR spectroscopy as they can selectively remove scalar and dipolar couplings and resonance overlap that cannot be removed otherwise by new pulse sequence designs. Dr. Alexander Koglin has extensive expertise in producing precursors for selective isotopic enrichment of MPs. For example, he used 1,3-13C2-glycerol and 2-13C1-glycerol as metabolic precursors to make a protein that is selectively isotopic enriched at 13C?, 13Cα, 2Hαand 15N positions, while the Cβposition is selectively 13C depleted and the adjacent protons are perdeuterated. This type of labeled protein allows the recording of HNCA spectrum with very high resolution in the Cαdimension by removing the 13Cα-13Cβcoupling. This is just one example of the power of labeling and there are many other labeling opportunities for solving difficult spectroscopic problems. Koglin is a faculty in the Bioscience division of Los Alamos National Laboratory (LANL). Previously, the B-8 of LANL/NIH stable isotope resource has worked out essentially all the synthetic pathways for the production of selectively isotopic enriched amino acids (AAs) and metabolic precursors. He will take advantage of the knowledge and expertise of this institution to produce metabolic precursors for all different types of isotopic enrichment. Using the resources of LANL, Koglin has developed novel procedures to synthesize AAs that are 13C, 2H and/or 15N labeled at almost any position to be desired (see Project 2). The labeled AAs are to be used with cell-free expression to avoid major scrambling. Some of these labeled AAs have already been used to determine the structure of the EntB module of Enterobactin Synthetase mega-enzyme (to be published). He will synthesize the AA precursors in LANL, as a collaborator of Wagner in Project 2. Then he will supervise a MP biochemist at Harvard Medical School (HMS) to test the precursors in producing selectively labeled MPs. This MP biochemist can then characterize the labeled MPs using the NMR facility at HMS. The precursors will be made available to other components of the Center through this Core. Some details of the planned synthesis are described in Appendix D. 3. Production of DNA nanotubes. As mentioned above in 1.5.2, the existing version of DNA nanotube for RDC measurement of MPs is not commercially available. Dr. William Shih in this Core will be responsible for producing the existing version of DNA nanotube to meet immediate demands in Project 1 ? 4. Since the production of this nanotube is labor intensive (i.e., amassing grams of single-stranded DNA using bacteriophage), Shih will recruit a full technician to perform this job. In addition, Shih will assist Chou in Project 1 to develop new versions of nanotubes (see description of the new developments in 1.5.2 above). As soon as the new nanotubes are developed, Shih will mass-produce them for distribution to other components of the Center.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54GM094608-04
Application #
8508952
Study Section
Special Emphasis Panel (ZGM1-CBB-3)
Project Start
Project End
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$400,606
Indirect Cost
$92,586
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Schlebach, Jonathan P; Sanders, Charles R (2015) Influence of Pathogenic Mutations on the Energetics of Translocon-Mediated Bilayer Integration of Transmembrane Helices. J Membr Biol 248:371-81
Yang, Qin; Bruschweiler, Sven; Chou, James J (2014) A self-sequestered calmodulin-like Caýýýýý sensor of mitochondrial SCaMC carrier and its implication to Caýýýýý-dependent ATP-Mg/P(i) transport. Structure 22:209-17
Roos, Christian; Kai, Lei; Haberstock, Stefan et al. (2014) High-level cell-free production of membrane proteins with nanodiscs. Methods Mol Biol 1118:109-30
OuYang, Bo; Chou, James J (2014) The minimalist architectures of viroporins and their therapeutic implications. Biochim Biophys Acta 1838:1058-67
Yang, Qin; Brüschweiler, Sven; Chou, James J (2014) Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/Pi carrier SCaMC1. Acta Crystallogr F Struct Biol Commun 70:68-71
Mittendorf, Kathleen F; Kroncke, Brett M; Meiler, Jens et al. (2014) The homology model of PMP22 suggests mutations resulting in peripheral neuropathy disrupt transmembrane helix packing. Biochemistry 53:6139-41
Song, Yuanli; Mittendorf, Kathleen F; Lu, Zhenwei et al. (2014) Impact of bilayer lipid composition on the structure and topology of the transmembrane amyloid precursor C99 protein. J Am Chem Soc 136:4093-6
Elter, Shantha; Raschle, Thomas; Arens, Sabine et al. (2014) The use of amphipols for NMR structural characterization of 7-TM proteins. J Membr Biol 247:957-64
Sun, Zhen-Yu J; Cheng, Yuxing; Kim, Mikyung et al. (2014) Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41. J Mol Biol 426:1095-108
Song, Yuanli; Kenworthy, Anne K; Sanders, Charles R (2014) Cholesterol as a co-solvent and a ligand for membrane proteins. Protein Sci 23:1-22

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