In order to understand how membrane proteins express their diverse biological functions, it is essential to determine their structures. However, membrane proteins present extraordinary technical difficulties for the most commonly used x-ray crystallography and NMR spectroscopy methods of structure determination, which were developed with water soluble globular proteins in mind. Alternative methods of protein structure determination are needed to analyze membrane proteins. The success of the research supported by this grant, especially in the development of a spectroscopic strategy invoking the powerful methods of high resolution solid-state NMR spectroscopy, enables us to propose structural studies of membrane proteins relevant to problems of medical urgency, including AIDS and heavy metal poisoning, and scientific importance, including the architecture of membrane proteins, membrane anchoring of proteins, protein processing, and modification of membrane properties by proteins. Although, experimental NMR studies of membrane proteins are near the limit of feasibility with currently available technology, we have demonstrated that by utilizing the most powerful spectroscopic experiments, spectrometers with the highest magnetic fields, and comprehensive isotopic labeling schemes, it is possible to obtain high resolution solid-state NMR and multidimensional solution NMR spectra of membrane proteins suitable for structural analyses. The overall goals of the research are to develop generally applicable NMR methods for determining the structures and describing the dynamics of membrane proteins and to apply these methods to membrane proteins that have interesting biological functions and are experimentally tractable. These methods will be applied to HIV-l Vpu protein, merP and merT proteins of the bacterial mercury detoxification system, and the coat and procoat proteins of filamentous bacteriophages. In addition, these methods will be applied in collaborative studies to the pulmonary surfactant proteins SP-B and SP-C and to subunit c of the F1F0 ATPase. These membrane proteins participate in biological processes not associated with globular proteins, are relatively small with between 35 and 116 residues, and can be prepared by expression in bacterial systems, automated solid-phase peptide synthesis, or both in substantial quantities specifically, selectively, or uniformly labeled with stable isotopes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020770-13
Application #
2442414
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-12-01
Project End
1998-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
13
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Kim, Y; Valentine, K; Opella, S J et al. (1998) Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers. Protein Sci 7:342-8
Marassi, F M; Ramamoorthy, A; Opella, S J (1997) Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers. Proc Natl Acad Sci U S A 94:8551-6
Almeida, F C; Opella, S J (1997) fd coat protein structure in membrane environments: structural dynamics of the loop between the hydrophobic trans-membrane helix and the amphipathic in-plane helix. J Mol Biol 270:481-95
Gesell, J; Zasloff, M; Opella, S J (1997) Two-dimensional 1H NMR experiments show that the 23-residue magainin antibiotic peptide is an alpha-helix in dodecylphosphocholine micelles, sodium dodecylsulfate micelles, and trifluoroethanol/water solution. J Biomol NMR 9:127-35
Almeida, F C; Opella, S J (1997) Measurement of 1H T1 rho in a uniformly 15N-labeled protein in solution with heteronuclear two-dimensional spectroscopy. J Magn Reson 124:509-11
Klassen, R B; Opella, S J (1997) NMR studies of peptides and proteins associated with membranes. Methods Mol Biol 60:271-97
Howard, K P; Opella, S J (1996) High-resolution solid-state NMR spectra of integral membrane proteins reconstituted into magnetically oriented phospholipid bilayers. J Magn Reson B 112:91-4
Bechinger, B; Gierasch, L M; Montal, M et al. (1996) Orientations of helical peptides in membrane bilayers by solid state NMR spectroscopy. Solid State Nucl Magn Reson 7:185-91
Tobias, D J; Gesell, J; Klein, M L et al. (1995) A simple protocol for identification of helical and mobile residues in membrane proteins. J Mol Biol 253:391-5
Opella, S J; Kim, Y; McDonnell, P (1994) Experimental nuclear magnetic resonance studies of membrane proteins. Methods Enzymol 239:536-60

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