We plan to use nuclear magnetic resonance spectroscopy (NMR) and computer methods to characterize protein structures in solution. Comparing the dynamic structures of wild-type and mutant proteins, we will develop NMR as a tool for protein molecular design. The work involves determining conformations of small proteins with 2D NMR experiments and distance geometry calculations and characterizing their dynamics by measuring hydrogen exchange rates and relaxation times, with many resonances as spatially assigned probes. Spin-spin coupling measurements will also be used to characterize the distribution of surface side-chain conformations. We will use site-directed mutagenesis in combination with NMR to investigate relationships between amino acid sequence, dynamic solution structure, and biological function. Because of its fundemental importance in protein molecular design, we will also study the kinetics and thermodynamics of protein folding. This will involve a search for local structures (folding nuclei or chain-folding initiation structures) in the """"""""unfolded state"""""""". In addition, we will try to find conditions where folding intermediates are selectively stabilized so that a spatial characterization by NMR of such metastable protein structures can be achieved. We will develop structural models of protein folding mechanisms based on these NMR data. Our initial investigations will focus on human transforming growth factor-alpha (TGF alpha) (MW=5000) and leech elastase inhibitor Eglin c (MW=7000). For both proteins we have access to the wild- type proteins and genetically engineered mutants produced in our collaborations with Dr. Michael Sporn (National Cancer Institute, NIH) and with Ciba-Geigy, Basel, Switzerland. Later on, we will investigate larger proteins such as transforming growth factor beta (TGF beta) (MW=22000), and other growth factors and oncogene proteins relevant in cancer research and pharamaceutical use. For the larger systems we will also employ techniques of biosynthetic enrichment for isotope labeling, in combination with the NMR work, for the purposes of simplifying the spectra and obtaining specific dynamic information. The immediate aim of this research is to obtain NMR-derived structural information on pharmaceutically important proteins and to develop experience using this information to correlate protein dynamic structure with function. The longer-range goals are to use such structural information to actually design proteins with preconceived biological and pharmaceutical properties, such as molecules which would alter the metabolism of malignant cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM038608-04
Application #
3295159
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1990-09-01
Project End
1993-03-31
Budget Start
1990-09-01
Budget End
1991-03-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Marintchev, Assen; Edmonds, Katherine A; Marintcheva, Boriana et al. (2009) Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation. Cell 136:447-60
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Roehrl, Michael H A; Wang, Julia Y; Wagner, Gerhard (2004) Discovery of small-molecule inhibitors of the NFAT--calcineurin interaction by competitive high-throughput fluorescence polarization screening. Biochemistry 43:16067-75
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Muller, Jens; Lugovskoy, Alexey A; Wagner, Gerhard et al. (2002) NMR structure of the [2Fe-2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase. Biochemistry 41:42-51
Zhou, P; Lugovskoy, A A; McCarty, J S et al. (2001) Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45. Proc Natl Acad Sci U S A 98:6051-5
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