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Article

Evaluation of Parameters Critical to Observing Proteins Inside Living Escherichia coli by In-Cell NMR Spectroscopy

Zach Serber,^† Richard Ledwidge,^‡ Susan M. Miller,^‡ and Volker Dötsch*^‡^§

Contribution from the Graduate Group in Biophysics, Department of Pharmaceutical Chemistry, Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94143

J. Am. Chem. Soc., 2001, 123 (37), pp 8895–8901

DOI: 10.1021/ja0112846

Publication Date (Web): August 16, 2001

†

Graduate Group in Biophysics.

‡

Department of Pharmaceutical Chemistry.

To whom correspondence should be addressed. Telephone: (415) 502-7050. Fax: (415) 476-0688. E-Mail: volker@picasso.ucsf.edu.

Department of Cellular and Molecular Pharmacology.

Abstract

Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of ¹⁵N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.

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History

Published In Issue September 19, 2001
Received May 25, 2001
Revised Manuscript Received July 6, 2001

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Article

Evaluation of Parameters Critical to Observing Proteins Inside Living Escherichia coli by In-Cell NMR Spectroscopy

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History

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Related Content

High-Resolution Macromolecular NMR Spectroscopy Inside Living Cells

Multidimensional NMR Spectroscopy for Protein Characterization and Assignment inside Cells

Methyl Groups as Probes for Proteins and Complexes in In-Cell NMR Experiments

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