The Use of Dansyl-Calmodulin to Study Interactions with Channels and Other Proteins
Steady-state fluorescence spectroscopy is a biophysical technique widely employed to characterize interactions between proteins in vitro. Only a few proteins naturally fluoresce in cells, but by covalently attaching fluorophores virtually all proteins ca
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Introduction Fluorescence spectroscopy is a widely used approach to analyze protein structure and function. Among the advantages of using fluorescence techniques are their high sensitivity, enabling minute amounts of sample material to be used, its noninvasive nature, the presence of natural intrinsic fluorophores in proteins, and the relatively simple equipment required to perform different experiments. These techniques are useful to study biochemical parameters, such as protein–protein interactions, conformational changes, metal binding, cellular localization, and much more. The intrinsic fluorescent probes in proteins are tyrosine and tryptophan residues, although the majority of studies using intrinsic protein fluorescence focus on tryptophan as it is almost always the dominant source of signal, and its fluorescence is much more sensitive to the environment than that of tyrosine. Unfortunately, there are usually only few tryptophan residues per protein, which means that the method senses only these few points in the proteins’ structure.
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_17, © Springer Science+Business Media, LLC 2013
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a N(CH3)2 NH C
O
NH NH2
+
C HCl
O C
O C
O S O
NH
N H
S O N(CH3)2
NH
Cl Lysine
Dansyl chloride
300
Excitation 350
Fluorescence Emission (Arbitrary Units)
Absorption (Arbitrary Units)
b
Emission 400 450 500 Wavelength (nm)
550
600
Fig. 1 Dansyl reactivity and its fluorescent properties. (a) Reaction scheme for the dansyl chloride labelling of a lysine residue. (b) Fluorescence emission spectrum of D-CaM (solid line, excitation at 340 nm) and excitation spectrum (dotted line, emission at 500 nm) in binding buffer
Furthermore, the signal is often weak, demanding the use of high protein concentrations. Thus, it is usually more convenient to use extrinsic fluorescence probes that can be covalently attached to a protein. There are many reagents available that generally react with amino or thiol groups, allowing the introduction of fluorophores that may detectably alter the spectral characteristics of the native protein. A commonly used fluorescent reagent is 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride or DNSchloride), which was for a long time the most widely used fluorogenic derivatizing reagent for amino acid determination in proteins and peptides (1). Dansyl chloride is a probe that binds to functional amine groups to form a fluorescent sulphonamide (Fig. 1a), but it can also react with the phenol group of tyrosine (2). The resulting dansyl derivates have proven particularly useful to analyze protein interactions since the dansyl emission spectrum is greatly perturbed by the local environment. In our laboratory, we have prepared dansyl-calmodulin (D-CaM) in order to characterize the interaction of calmodulin (CaM) with different components of the Kv7.2 channel CaM binding domain (Kv7.2 CBD). Since the intensity of the D-CaM em
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