The Muir and Cowburn laboratories propose to test definitively the hypothesis that segmental isotopic labeling using expressed protein ligation can extend significantly the range of targets amenable to structural studies using NMR. Having demonstrated the practicality of this approach recently, it is proposed to extend the procedure to permit selective labeling of internal segments of a protein, to improve significantly the efficiency and practicality of the methods with a view to making them routine, and to developing these approaches with a range of significant target systems, including the src homology adaptor, Crk, the restriction endonuclease, TaqI, the GTPase effector domain, Dbl homology in conjunction with the sequentially associated Pleckstrin homology domain, and scaffold/adaptors in the MAP kinase pathways. These technological improvements will include the selection of better ways to prepare recombinant polypeptide segments containing the necessary chemically reactive groups for expressed protein ligation, as well as the development of a solid-phase expressed protein ligation strategy which will allow multiple polypeptide segments to be efficiently and quickly pieced together. The development of the segmental isotopic labeling methods will significantly broaden the applications of expressed protein ligation, specifically with regard to NMR, so that structural studies in solution can, in combination with other technological developments in NMR, apply to a much more significant range of molecular structural problems in health related biology. Examples directly connected to the project may lead to improvements in recombinant DNA technology by increased understanding of restriction enzymes, deeper insight into mechanisms of signal transduction involving normal development and homeostasis, as well as disorders associated with infection, cancer, and aging.
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| Mootz, Henning D; Blum, Elyse S; Tyszkiewicz, Amy B et al. (2003) Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 125:10561-9 |
| Wilson, Kelly-Anne; Kalkum, Markus; Ottesen, Jennifer et al. (2003) Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. J Am Chem Soc 125:12475-83 |
| Shekhtman, Alexander; Ghose, Ranajeet; Goger, Michael et al. (2002) NMR structure determination and investigation using a reduced proton (REDPRO) labeling strategy for proteins. FEBS Lett 524:177-82 |
| Camarero, Julio A; Shekhtman, Alexander; Campbell, Elizabeth A et al. (2002) Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci U S A 99:8536-41 |
| Mootz, Henning D; Muir, Tom W (2002) Protein splicing triggered by a small molecule. J Am Chem Soc 124:9044-5 |
| Cowburn, D; Muir, T W (2001) Segmental isotopic labeling using expressed protein ligation. Methods Enzymol 339:41-54 |
