Abstract
A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-1232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects. This finding supports the proposal that the face of the central helix containing lysine 75 is involved in interaction with MLC kinase and suggests also that additional contact near Ca2-binding site 1 occurs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 202-209 |
| Number of pages | 8 |
| Journal | Proteins: Structure, Function and Genetics |
| Volume | 2 |
| Issue number | 3 |
| State | Published - 1987 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Genetics
- Structural Biology
Cite this
Association of calmodulin and smooth muscle myosin light chain kinase : Application of a label selection technique with trace acetylated calmodulin. / Jackson, A. E.; Carraway, Kermit L; Payne, M. E.; Means, A. R.; Puett, D.; Brew, K.
In: Proteins: Structure, Function and Genetics, Vol. 2, No. 3, 1987, p. 202-209.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Association of calmodulin and smooth muscle myosin light chain kinase
T2 - Application of a label selection technique with trace acetylated calmodulin
AU - Jackson, A. E.
AU - Carraway, Kermit L
AU - Payne, M. E.
AU - Means, A. R.
AU - Puett, D.
AU - Brew, K.
PY - 1987
Y1 - 1987
N2 - A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-1232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects. This finding supports the proposal that the face of the central helix containing lysine 75 is involved in interaction with MLC kinase and suggests also that additional contact near Ca2-binding site 1 occurs.
AB - A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-1232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects. This finding supports the proposal that the face of the central helix containing lysine 75 is involved in interaction with MLC kinase and suggests also that additional contact near Ca2-binding site 1 occurs.
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UR - http://www.scopus.com/inward/citedby.url?scp=0023632423&partnerID=8YFLogxK
M3 - Article
C2 - 3128785
AN - SCOPUS:0023632423
VL - 2
SP - 202
EP - 209
JO - Proteins: Structure, Function and Bioinformatics
JF - Proteins: Structure, Function and Bioinformatics
SN - 0887-3585
IS - 3
ER -