Studying the Spatial Distribution of Ca2+-Binding Proteins

How Does it Work for Calmodulin?
  • Katalin Török
  • Richard Thorogate
  • Steven Howell
Part of the Methods in Molecular Biology™ book series (MIMB, volume 173)

Abstract

Calmodulin is a ubiquitous Ca2+-switch protein whose in vitro properties have been widely studied (1). Visualization of calmodulin levels and functional changes in living cells allows investigations of how calmodulin is involved in organizing specific cellular responses to various stimuli. The advancement of several protein chemistry, biochemical, and microscopic techniques has made the direct study of calmodulin in cellular function less perturbing, more sensitive, and of higher temporal and spatial resolution. For example, more selective fluorescent-labeling techniques directed at strategically positioned Lys and Cys residues in the protein are now available, the latter are introduced by site-directed mutagenesis. In addition, the conjugation of calmodulin c-DNA with enhanced green fluorescent protein (GFP) provides increased sensitivity. Taken together, these advances allow the fluorescence signal of the protein to act as an intracellular reporter group of concentration changes in cell compartments, as well as other well-defined molecular events (e.g., Ca2+and target binding, conformational change). Brighter fluorophores provide increased sensitivity and thus the fluorescent protein may be applied at a lower concentration to act as a tracer of endogenous calmodulin. Calmodulins with probes attached at a single site that have been characterized functionally by comparison to unmodified calmodulin, facilitate clearer data interpretation. Laser-scanning confocal microscopy offers higher resolution so events in living cells can be monitored in greater detail in order to understand calmodulin and its interactions in the cell.

Keywords

HPLC Chlorine Syringe Lysine CaCl2 

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Katalin Török
    • 1
  • Richard Thorogate
    • 1
  • Steven Howell
    • 2
  1. 1.Department of Pharmacology and Clinical PharmacologySt. George’s Hospital Medical SchoolLondonUK
  2. 2.National Institute for Medical ResearchLondonUK

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