Studying the Spatial Distribution of Ca2+-Binding Proteins
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.
KeywordsHPLC Chlorine Syringe Lysine CaCl2
- 1.Cohen, P. and Klee, C. B., eds. (1988) Calmodulin. Elsevier, New York.Google Scholar
- 9.Allen, G. (1989) Sequencing of proteins and peptides. Laboratory techniques in Biochemistry and Molecular Biology (Burdon, R. H. and van Knippenberg, eds.), Elsevier, Amsterdam.Google Scholar
- 11.Cowley, D. J., O’Kane, E., and Todd, R. S. J. (1991) Triazinylaniline derivatives as fluorescence probes. Part 1. Absorption and fluorescence in organic solvents and in aqueous media in relation to twisted intramolecular charge-transfer state formation, H bonding and protic equilibria. J. Chem. Soc. Perkin. Trans. 2, 1495–1500.Google Scholar
- 13.Török, K., Cowley, D. J., Brandmeier, B. D., Howell, S., Aitken A., and Trentham. D. R. (1998) Inhibition of calmodulin-activated smooth muscle myosin light chain kinase by calmodulin binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives. Biochemistry 37, 6188–6198.PubMedCrossRefGoogle Scholar
- 16.Pawley, J., ed. (1989) The Handbook of Biological Confocal Microscopy. IMR Press, Madison, Wisconsin.Google Scholar