We continue to make progress on the development of new solid state nuclear magnetic resonance (NMR) methods for structural studies of peptides and proteins, and on the application of these methods to specific systems of biological or biomedical importance. Progress in methodological development in FY2001 includes: (1) The development of a new solid state NMR technique, called DQCSA spectroscopy, that permits the determination of a peptide or protein backbone conformation at a specific doubly-13C-labeled site. This technique has been successfully demonstrated on model peptides and proteins of known structure, and has been applied in structural studies of Alzheimer's beta-amyloid fibrils and HIV-1 Rev protein fibrils; (2) The development of a new solid state NMR technique, called fpRFDR-CT spectroscopy, that permits the measurement of interatomic distances in selectively 13C-labeled peptides and proteins. This technique has been used to determine the structural organization and extent of beta-sheets in Alzheimer's beta-amyloid fibrils; (3) The development of new methods for sensitivity enhancement in solid state NMR measurements. These methods include proton-detected 13C and 15N NMR and pulsed-spin-lock detection in 13C NMR. Both methods provide sensitivity enhancements by factors of 3 to 10, making measurements possible that would otherwise be prohibited by inavailability of adequate quantities of labeled peptides or proteins. Progress in applications to specific systems includes: (1) Further characterization of the molecular structures of Alzheimer's beta-amyloid fibrils, including the identification of structurally ordered and structurally disordered regions, identification of beta-strand segments, and identification of non-beta-strand conformations at specific sites. Results so far suggest that full molecular structure determination by solid state NMR will soon be possible, and this is the main goal of current research efforts; (2) Structural studies of the HIV-1 Rev protein in fibrillar form. Measurements of the protein backbone conformation at specific sites support a helix-loop-helix structural model for the N-terminal half of HIV-1 Rev. These are the first atomic-level structural constraints on the full-length Rev protein, which has not been amenable to structure determination by more traditional liquid state NMR or x-ray crystallographic methods; (3) Development of protocols for synthesis, purification, disulfide bridge formation, and fibrillization of the full-length amylin peptide (a.k.a. islet amyloid precursor protein) associated with type 2 diabetes. This work sets the stage for structural measurements on amylin fibrils analogous to our work on Alzheimer's beta-amyloid fibrils.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK029029-05
Application #
6508984
Study Section
(LCP)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2001
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Qiang, Wei (2011) Signal enhancement for the sensitivity-limited solid state NMR experiments using a continuous, non-uniform acquisition scheme. J Magn Reson 213:171-5
Hu, Kan-Nian; Tycko, Robert (2009) Zero-quantum frequency-selective recoupling of homonuclear dipole-dipole interactions in solid state nuclear magnetic resonance. J Chem Phys 131:045101
Tycko, Robert (2007) Stochastic dipolar recoupling in nuclear magnetic resonance of solids. Phys Rev Lett 99:187601
Tycko, Robert (2007) Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR. J Chem Phys 126:064506
Paravastu, Anant K; Petkova, Aneta T; Tycko, Robert (2006) Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide. Biophys J 90:4618-29
Tycko, Robert (2006) Molecular structure of amyloid fibrils: insights from solid-state NMR. Q Rev Biophys 39:1-55
Tycko, Robert (2006) Solid-state NMR as a probe of amyloid structure. Protein Pept Lett 13:229-34
Sharpe, Simon; Yau, Wai-Ming; Tycko, Robert (2006) Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning. Biochemistry 45:918-33
Petkova, Aneta T; Yau, Wai-Ming; Tycko, Robert (2006) Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry 45:498-512
Tycko, Robert (2006) Characterization of amyloid structures at the molecular level by solid state nuclear magnetic resonance spectroscopy. Methods Enzymol 413:103-22

Showing the most recent 10 out of 42 publications