Solution conformations of the N-terminal CNBr fragment of glycogen phosphorylase and its interaction with calmodulin.
The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase.
Takrama
J F
JF
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Graves
D J
DJ
D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000494:pharmacology; D005453:Fluorescence; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry*; D006005:Phosphorylases / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D012996:Solutions
D002147:Calmodulin; D010446:Peptide Fragments; D012996:Solutions; D006005:Phosphorylases; D003488:Cyanogen Bromide; D002118:Calcium