The long-term objectives are to develop and apply state-of-the-art NMR techniques to the determination of the solution structural properties of a ubiquitously distributed protein involved in metal homeostasis, metallothioneins, MTs. This information is essential for the elucidation of the molecular mechanism of action of MT in metal regulation. An integral component of the proposed research is to contribute the adaptation/refinement of novel new NMR methods for macromolecular studies. While detailed structural information has recently become available through NMR and X-ray crystallographic methods for the Cd, Zn containing mammalian MT, similar structural information is not available for the Cu+ containing forms of the mammalian MT, nor for the MTs from other species (Saccharomyces cerevisiae, Neurospora crassa). Specific NMR experiments are proposed using the Ag+ ion as a chemically and magnetically (spin I=1/2) isomorphic metal ion substitute for Cu+ for the determination of the solution structure of this metal form of MT. NMR methods will also be used for the full solution structure determination of MTs from the marine crustaceans, the American lobster and the blue crab, which bind the divalent metal ions, Cd and Zn, and/or the monovalent metal ion, Cu. Selected MT samples from these sources, which will include recombinant forms of Chinese hamster MT, will concurrently and/or subsequently be used in experiments whose objectives are to delineate the kinetics and mechanistic features associated with this protein's physiological role in metal homeostasis. It is tempting to speculate that because of the analogies of the metal- binding motifs in both the transcription factors and MT, that the latter could have a metalloregulatory function in cellular repair processes, growth, and differentiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018778-18
Application #
2137224
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-05-01
Project End
1995-08-31
Budget Start
1994-04-01
Budget End
1995-08-31
Support Year
18
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Cobine, Paul A; McKay, Ryan T; Zangger, Klaus et al. (2004) Solution structure of Cu6 metallothionein from the fungus Neurospora crassa. Eur J Biochem 271:4213-21
Zangger, Klaus; Armitage, Ian M (2002) Dynamics of interdomain and intermolecular interactions in mammalian metallothioneins. J Inorg Biochem 88:135-43
Oz, G; Zangger, K; Armitage, I M (2001) Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3. Biochemistry 40:11433-41
Zangger, K; Shen, G; Oz, G et al. (2001) Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem J 359:353-60
Zangger, K; Oz, G; Armitage, I M (2000) Re-evaluation of the binding of ATP to metallothionein. J Biol Chem 275:7534-8
Zangger, K; Oz, G; Otvos, J D et al. (1999) Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy. Protein Sci 8:2630-8
Zangger, K; Armitage, I M (1998) Sensitivity-enhanced detection of fast exchanging protons by an exchange-edited gradient HEHAHA-HSQC experiment. J Magn Reson 135:70-5
Peterson, C W; Narula, S S; Armitage, I M (1996) 3D solution structure of copper and silver-substituted yeast metallothioneins. FEBS Lett 379:85-93
Okar, D A; Kakalis, L T; Narula, S S et al. (1995) Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy. Biochem J 308 ( Pt 1):189-95
Narula, S S; Brouwer, M; Hua, Y et al. (1995) Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy. Biochemistry 34:620-31

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