Predicted calmodulin‐binding sequence in the γ subunit of phosphorylase b kinase

A basic, amphiphilic α helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin‐binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin‐binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (γ) subunit of phosphorylase b kinase for interaction with calmodulin (the δ subunit). A peptide corresponding to this site (residues 341–361 of the γ subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the γ subunit) was replaced with an asparagin was found to bind calmodulin with approximately a 3 nM dissociation constant.

[1]  P. Cohen,et al.  Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits. , 1980, European journal of biochemistry.

[2]  A. Edelman,et al.  Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase. , 1985, The Journal of biological chemistry.

[3]  Y H Chen,et al.  Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. , 1974, Biochemistry.

[4]  M. Whitlow,et al.  Energy minimization for tertiary structure prediction of homologous proteins: alpha 1-purothionin and viscotoxin A3 models from crambin. , 1985, Journal of biomolecular structure & dynamics.

[5]  David Eisenberg,et al.  The helical hydrophobic moment: a measure of the amphiphilicity of a helix , 1982, Nature.

[6]  K. F. Chan,et al.  Isolation and properties of the catalytically active gamma subunit of phosphorylase b kinase. , 1980, The Journal of biological chemistry.

[7]  W. DeGrado,et al.  The interaction of calmodulin with amphiphilic peptides. , 1985, The Journal of biological chemistry.

[8]  E. Krebs,et al.  Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase. , 1986, Biochemistry.

[9]  P. Cohen,et al.  Identification of the Ca2+‐dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase , 1978, FEBS letters.

[10]  G. Fasman,et al.  Computed circular dichroism spectra for the evaluation of protein conformation. , 1969, Biochemistry.

[11]  R. Hodges,et al.  Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]. , 1986, Biochemistry.

[12]  W. DeGrado,et al.  p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide. , 1986, The Journal of biological chemistry.

[13]  P. Cohen,et al.  Activation of phosphorylase kinase from rabbit skeletal muscle by calmodulin and troponin , 1979, FEBS letters.

[14]  W. DeGrado,et al.  The design, synthesis, and characterization of tight‐binding inhibitors of calmodulin , 1985, Journal of cellular biochemistry.

[15]  P. Argos.  Protein structural models for nucleic acid interactions. , 1981, Journal of theoretical biology.

[16]  R. Doolittle.  Similar amino acid sequences: chance or common ancestry? , 1981, Science.

[17]  K. F. Chan,et al.  Isolation and physicochemical properties of active complexes of rabbit muscle phosphorylase kinase. , 1982, The Journal of biological chemistry.

[18]  P. Y. Chou,et al.  Empirical predictions of protein conformation. , 1978, Annual review of biochemistry.

[19]  A. Edelman,et al.  Amino acid sequence of an active fragment of rabbit skeletal muscle myosin light chain kinase. , 1985, Biochemistry.

[20]  An SN2 deprotection of synthetic peptides with a low concentration of hydrofluoric acid in dimethyl sulfide: evidence and application in peptide synthesis , 1983 .

[21]  R. D. Wade,et al.  Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. , 1984, Biochemistry.

[22]  D. Blumenthal,et al.  Interaction of calmodulin and a calmodulin-binding peptide from myosin light chain kinase: major spectral changes in both occur as the result of complex formation. , 1985, Biochemistry.

[23]  P. Cohen,et al.  The role of calcium ions, calmodulin and troponin in the regulation of phosphorylase kinase from rabbit skeletal muscle. , 1980, European journal of biochemistry.

[24]  F G Prendergast,et al.  Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase. , 1986, Biochemistry.

[25]  A. Edelman,et al.  Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Moras,et al.  Three-dimensional structure of D-glyceraldehyde-3-phosphate dehydrogenase. , 1974, Journal of molecular biology.

[27]  D. Malencik,et al.  Peptide binding by calmodulin and its proteolytic fragments and by troponin C. , 1984, Biochemistry.

[28]  G. Sanyal,et al.  Probable role of amphiphilicity in the binding of mastoparan to calmodulin. , 1985, Biochemistry.

[29]  D. Blumenthal,et al.  Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase. , 1982, Biochemistry.

[30]  R. D. Wade,et al.  Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase. , 1984, Biochemistry.

[31]  W. DeGrado,et al.  [37] Recognition and characterization of calmodulin-binding sequences in peptides and proteins , 1987 .

[32]  D. Graves,et al.  Isolation and properties of the active gamma subunit of phosphorylase kinase. , 1986, The Journal of biological chemistry.

[33]  C. Klee,et al.  [27] Purification of smooth muscle myosin light-chain kinase , 1982 .

[34]  D. Eisenberg,et al.  Analysis of membrane and surface protein sequences with the hydrophobic moment plot. , 1984, Journal of molecular biology.