Figure 1

Interaction between human brain MBP and CaM and selection of the peptide for the CaM interaction study. A. Electrophoretic analysis by SDS-PAGE of the fractions from affinity chromatography of human brain MBP on CaM-sepharose. The samples are as follows: 1, adult human brain MBP isoforms loaded into the column; 2–6, washes with a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM CaCl₂; 7–11, elution with a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM EGTA. The observed strong protein band corresponds to the 18.5-kDa main human MBP isoform. The presence of also the 17.2-kDa isoform in both the second wash (lane 3) and the second EGTA eluate (lane 8) was confirmed by mass spectrometry (see Additional file 1). B. The sequence of human 18.5-kDa MBP, in which the bold, boxed region is the peptide eventually selected. Below the sequence, various results from sequence analysis are shown: NPSA secondary structure prediction, black arrows (helix) and cylinders (strand); DISembl disorder prediction using the 3 different learning sets in the program, blue-red-green; Wiggle prediction for functionally flexible regions, magenta; GlobPlot prediction of disorder, orange; suggested CaM-binding sites according to the CaM target database, yellow; DRIP-PRED disorder prediction, black. C. Manual alignment of the selected peptide with CaM-binding segments from three human CaM-dependent protein kinases. The positions of the conserved hydrophobic anchor residues in CaM-dependent kinases are indicated below the alignment.