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In-cell NMR spectroscopy.

Zach Serber1, Lorenzo Corsini, Florian Durst

  • 1Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305, USA.

Methods in Enzymology
|April 6, 2005
PubMed
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Nuclear Magnetic Resonance (NMR) spectroscopy enables detailed analysis of protein conformation and dynamics within living cells. In-cell NMR techniques are presented, highlighting labeling strategies and current detection limits for protein studies.

Area of Science:

  • Biophysics
  • Structural Biology
  • Cellular Biology

Background:

  • Protein function is dictated by cellular location and three-dimensional conformation.
  • Fluorescence techniques excel at protein localization in vivo but lack conformational detail.
  • A gap exists in analyzing protein structure and dynamics within the cellular environment.

Purpose of the Study:

  • To describe technical aspects of Nuclear Magnetic Resonance (NMR) spectroscopy for in-cell experiments.
  • To investigate the feasibility and limitations of "in-cell NMR" for protein analysis.
  • To assess the impact of labeling strategies, background noise, and cellular viscosity on in-cell NMR.

Main Methods:

  • Utilized Nuclear Magnetic Resonance (NMR) spectroscopy for in-cell experiments.

Related Experiment Videos

  • Investigated (15)N and (13)C labeling schemes to assess background noise.
  • Correlated signal-to-noise ratios with protein overexpression levels to determine detection limits.

    • Main Results:

    • (15)N-labeling schemes show negligible background noise in in-cell NMR.
    • (13)C-based experiments require selective labeling due to high background.
    • The current detection limit for in-cell NMR is 150-200 µM (intracellular concentration).
    • Intracellular viscosity is not a limiting factor, with rotational correlation times only ~2x longer than in water.

    Conclusions:

    • In-cell NMR spectroscopy is a viable technique for studying protein conformation and dynamics in living cells.
    • Optimization of labeling strategies is crucial for successful in-cell NMR, particularly for (13)C experiments.
    • The technique's sensitivity and the influence of the cellular environment have been characterized.