title: In-cell NMR spectroscopy.
abstract: The role of a protein inside a cell is determined by both its location and its conformational state. Although fluorescence techniques are widely used to determine the cellular localization of proteins in vivo, these approaches cannot provide detailed information about a protein's three-dimensional state. This gap, however, can be filled by NMR spectroscopy, which can be used to investigate both the conformation as well as the dynamics of proteins inside living cells. In this chapter we describe technical aspects of these "in-cell NMR" experiments. In particular, we show that in the case of (15)N-labeling schemes the background caused by labeling all cellular components is negligible, while (13)C-based experiments suffer from high background levels and require selective labeling schemes. A correlation between the signal-to-noise ratio of in-cell NMR experiments with the overexpression level of the protein shows that the current detection limit is 150-200 muM (intracellular concentration). We also discuss experiments that demonstrate that the intracellular viscosity is not a limiting factor since the intracellular rotational correlation time is only approximately two times longer than the correlation time in water. Furthermore, we describe applications of the technique and discuss its limitations.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305, USA.
authors.forename: Zach
authors.initials: Z
authors.lastname: Serber
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Corsini
authors.forename: Florian
authors.initials: F
authors.lastname: Durst
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002147:Calmodulin; D009587:Nitrogen Isotopes; D011506:Proteins