 Leonard SpicerUniversity Distinguished Service Professor of Radiology and Professor of BiochemistryStructure-function studies of proteins using high-field NMR techniques; physical biochemistry. Contact InformationOffice Number: (919) 684-4327 Fax: (919) 684-8885 e-mail spicer@biochem.duke.edu Lab Location 235A Nanaline Duke Building Mailing Address Department of Biochemistry Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 Education
Research InterestsMacromolecular structure and dynamics often play critical roles in determining biological function. We are using physical biochemical approaches to characterize proteins and their functional assemblies involved in repressor activity in E. coli and in human DNA repair by nucleotide excision. We are also pursing collaborative projects in HIV vaccine development based on structural determinants associated with the C4 and V3 domains of the HIV-1 coat protein, gp120. One of the primary methods we use is ultra high field NMR spectroscopy, and we have installed state-of-the-art spectrometers operating at 800 MHz, 600 MHz, and 500 MHz for this purpose in the NMR Center. The MetJ repressor is a methionine regulatory protein in E. coli. The repressor complex operates by assembling two or more protein dimers on tandem 8 base pair DNA recognition sites of the met regulon in the presence of the downstream co-repressor S- adenosylmethionine, (SAM). While the structures of the protein dimer and tetramer-DNA-SAM complex are known, they reveal little about the mechanism of MetJ activation by SAM and the individual processes of initial DNA-binding and protein-protein recruitment, key factors in repressor function. NMR results are providing unique insights into the pathway by which this transcription factor operates. We are also studying the structural determinants in recognition and repair of DNA via the human nucleotide excision repair mechanism. A structure for the minimal DNA-binding domain of the human XPA protein has been determined by NMR in a collaborative effort and the DNA-binding site as well as sites of protein-protein interaction with other components in the multiprotein repair assembly are being probed. The goal is to understand the detailed interplay of protein dynamics with the mechanistic steps by which the several proteins bind DNA lesions and interact with one another in carrying out the important biological repair function Recent Publications1. A.M. Augustus, P.N. Reardon, W.T. Heller, and L.D. Spicer, Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ (2006) J. Biological Chem. 281 34269-34276 More… 2. P.N. Reardon and L.D. Spicer (2005) “Multidimensional NMR Spectroscopy for Protein Characterization and Assignment inside Cells.” J. Am. Chem. Soc. 127:10848-10849. More… 3. R. Sinclair, L.D. Spicer, et al. (2005), Midsize Facilities: Infrastructure for Materials Research, The National Academies Press, Washington, D.C. pps. 1-250. More… 4. G.W. Buchko, N.G. Isern, L.D. Spicer* and M.A. Kennedy* (2001) “Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219).” Mutation Research 486:1-10. More… 5. G.W. Buchko, C-S Tung, K. McAteer, N.G. Isern, L.D. Spicer* and M.A. Kennedy* (2001) “DNA-XPA Interactions: A 31P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA binding domain (M98-F219) of the nucleotide excision repair protein XPA.” Nucleic Acid Research 29:2635-43. More… 6. H.M. Vu, D. Myers, R. de Lorimier, T. Matthews, M. Moody, C. Heinly, J. Torres, B. Haynes and L. Spicer (1999) “NMR analysis of solution conformation in C4-V3 hybrid peptides derived from HIV-1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies.” J. Virology 73:746-50. More… 7. G.W. Buchko, G.W. Daughdrill, R. de Lorimier, B.K. Sudha Rao, N. G. Isern, J. Lingbeck, J. Taylor, M.S. Wold, M. Gochin, L.D. Spicer, D.F. Lowry and M.A. Kennedy*. (1999) “Interactions of human nucleotide excision repair protein XPA with DNA and RPA70ÆC327: chemical shift mapping and 15N NMR relaxation studies.” Biochemistry 38:15116-28. More… 8. R.A. Venters, H.M. Vu, R. M. de Lorimier and L.D. Spicer (1997) “Strategies for NMR assignment and global fold determinations using perdeuterated proteins.” Techniques in Protein Chemistry VIII, pps. 605-615. More… 9. R. De Lorimier, H. W. Hellinga and L. D. Spicer (1996) “NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.” Protein Sci 12:2552-65. More… 10. R. A. Venters, B. T. Farmer, 2nd, C. A. Fierke and L. D. Spicer (1996) “Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II.” J Mol Biol 5:1101-16. More… 11. D. E. Hyre and L. D. Spicer (1995) “Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry.” Biochemistry 10:3212-21. More… 12. R. A. Venters, C. C. Huang, B. T. Farmer, 2nd, R. Trolard, L. D. Spicer and C. A. Fierke (1995) “High-level 2H/13C/15N labeling of proteins for NMR studies.” J Biomol NMR 4:339-44. More… |
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