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Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Intracellular Protien NMR

The recent development of ?in-cell NMR? techniques has demonstrated that NMR spectroscopy can be used to characterize the conformation and dynamics of biological macromolecules inside living cells. We show that labeling of proteins with 15N can be achieved with minimal background from bacterial proteins. We compare several different protocols and media and demonstrate that the overexpression level is the most critical parameter. Virtually background-free in-cell NMR spectra, however, can be obtained by amino-acid type selective labeling. We further demonstrate that this selective labeling strategy can also be used for very efficient backbone assignment for in vitro samples. In addition we present new labeling procedures for 13C-labeling of selective proteins in the cytoplasm which can overcome the problem of high background from cellular molecules. This labeling strategy can also be used for studying large proteins in the bacterial cytoplasm. Finally, we will present applications of these in-cell NMR labeling techniques that include investigation of the dynamics of proteins and drug-protein interactions in the cytoplasm. In these experiments we demonstrate that differences in the dynamics between the intracellular and the in vitro forms of proteins can exist. In particular, backbone nitrogen spins of the metal binding loop of the small bacterial protein NmerA relax faster inside the bacterial cytoplasm than in an in vitro sample. We will discuss models that can explain these results.

Volker Doetsch

Dept. Pharmaceutical Chemistry
UCSF
San Francisco, CA 94143
volker@picasso.ucsf.edu