Identification of calmodulin-binding proteins

Melvin Billingsley, Joseph W. Polli, Keith R. Pennypacker, Randall L. Kincaid

Research output: Contribution to journalArticle

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Abstract

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic maping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to authoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphoriesterase occurs, but this interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin.15 One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin.30 The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Original languageEnglish (US)
Pages (from-to)451-467
Number of pages17
JournalMethods in Enzymology
Volume184
Issue numberC
DOIs
StatePublished - Jan 1 1990

Fingerprint

Calmodulin-Binding Proteins
Calmodulin
Proteins
Biotinylation
Derivatives
Type 1 Cyclic Nucleotide Phosphodiesterases
Amino Acids
Chromogenics
Acetylation
Avidin
Biotin
Radioactivity
Phosphoric Diester Hydrolases
Chemical modification
Yeast
Carrier Proteins
Yeasts

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Billingsley, M., Polli, J. W., Pennypacker, K. R., & Kincaid, R. L. (1990). Identification of calmodulin-binding proteins. Methods in Enzymology, 184(C), 451-467. https://doi.org/10.1016/0076-6879(90)84307-3
Billingsley, Melvin ; Polli, Joseph W. ; Pennypacker, Keith R. ; Kincaid, Randall L. / Identification of calmodulin-binding proteins. In: Methods in Enzymology. 1990 ; Vol. 184, No. C. pp. 451-467.
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Billingsley, M, Polli, JW, Pennypacker, KR & Kincaid, RL 1990, 'Identification of calmodulin-binding proteins', Methods in Enzymology, vol. 184, no. C, pp. 451-467. https://doi.org/10.1016/0076-6879(90)84307-3

Identification of calmodulin-binding proteins. / Billingsley, Melvin; Polli, Joseph W.; Pennypacker, Keith R.; Kincaid, Randall L.

In: Methods in Enzymology, Vol. 184, No. C, 01.01.1990, p. 451-467.

Research output: Contribution to journalArticle

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N2 - We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic maping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to authoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphoriesterase occurs, but this interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin.15 One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin.30 The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

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Billingsley M, Polli JW, Pennypacker KR, Kincaid RL. Identification of calmodulin-binding proteins. Methods in Enzymology. 1990 Jan 1;184(C):451-467. https://doi.org/10.1016/0076-6879(90)84307-3