Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria

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Abstract

The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca2+-ATPase was overexpressed in Escherichia coli as a 48 kDa fusion protein, designated p48, containing an N-terminal hexa-His tag. Purification conditions were optimized, thus conferring long-term stability to p48. Circular dichroism spectroscopy and the pattern of limited trypsinolysis confirmed the proper folding of the domain. p48 retained 0.5 ± 0.1 mol of high affinity 2’,3’-O-(2,4,6-trinitrophenyl)adenosine-5’-triphosphate (TNP-ATP) binding sites per mol of polypeptide chain with an apparent dissociation constant of about 8 µM. Size-exclusion FPLC using protein concentrations in the range 0.03-5 mg/ml showed that p48 was essentially monodisperse with apparent molecular mass and Stokes radius (Rs) values compatible with a dimer (100 kDa and 40 Å, respectively). Analysis of p48 by small-angle X-ray scattering provided an independent second proof for a dimeric p48 particle with a radius of gyration (Rg) of 39 Å, suggesting that the dimer was not spherical (Rs/Rg=1.026). When digested by proteinase K, p48 was converted to a 30 kDa fragment, designated p30, which was very resistant to further proteolysis. p30 retained high affinity TNP-ATP binding (Kd = 8 µM) and eluted as a monomer (35 kDa) in size-exclusion FPLC. As opposed to p48, the p30 fragment did not react with monoclonal antibody A52 [Clarke et al., J. Biol. Chem. 264 (1989) 11246-11251] which recognizes region E657-R672 located upstream of the hinge domain of the Ca2+-ATPase. These results indicate a requirement of the hinge domain (670-728) region for self-association of the p48 large hydrophilic domain as a dimer. We propose that this behavior points to a possible role of the hinge domain in dimerization of sarcoplasmic reticulum Ca2+-ATPase in the native membrane. © 2000 Elsevier Science B.V.

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Carvalho-Alves, P. C., Hering, V. R., Oliveira, J. M. S., Salinas, R. K., & Verjovski-Almeida, S. (2000). Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria. Biochimica et Biophysica Acta - Biomembranes, 1467(1), 73–84. https://doi.org/10.1016/S0005-2736(00)00206-6

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