Physicochemical and hydrodynamic characterization of P-57, a neurospecific calmodulin binding protein

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Abstract
P-57 is a neurospecific calmodulin bidning protein that was discovered by virtue of its unusual interactions with calmodulin-Separose [Andreason, T. J., Leutje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreason, T. J., Andreason, K. I., and Storm, D. R. (1985) J. Biol. Chem. 260, 10784-10788]. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin-Sepharose in the absence of calcium compared to that in the presence of calcium. In this study, we report the chemical and physical properties of P-57 purified from detergent-solubilized bovine brain membranes. The amino acid composition of P-57 is distinctive in that it contains a single phenylalanine residue with no other aromatic amino acids and a relatively high percentage of proline and alanine. In the presence of 0.05% Lubrol PX, its predicted secondary structure from circular dichroism spectroscopy is 1% .alpha.-helix, 21% .beta.-sheet, and 78% random coil. The hydrodynamic characteristics of the protein-detergent complex and the molecular weight of the protein-detergent complex and the molecular weight of the protein were determined by gel filtration and sucrose density gradient sedimentation in the presence and absence of calmodulin. The P57-detergent complex has an apparent Stokes radius (Rs) of 4.58 nm and a sedimentation coefficient (S20,w) of 1.44 S while the Stokes radius and S20,w for the P-57-calmodulin-detergent complex are 5.33 nm and 2.32 S respectively. Perrin analysis of a 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid (AEDANS) derivative of P-57 confirmed the Stokes radius determined by gel filtration. Frictional coefficients of 1.88 and 1.83 for the P-57-detergent and P-57-calmodulin-detergent complexes suggest elongated asymmetric particles. Partial specific volumes of 0.778 and 0.798 mL/g corresponding to molecular weights of 36 800 and 63 213 were obtained for the P-57-detergent and the P-57-calmodulin-detergent complexes, respectively. The estimated molecular weights for P-57 and the P-57-calmodulin complex without detergent are 25 700 and 45 600, respectively, although P-57 runs with an apparent Mr of 54 100 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. These data indicate that P-57 is an elongated calmodulin binding protein with an unusual amino acid composition and atypical behavior on SDS gel electrophoresis.