Abstract
Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).
Highlights
Altered calmodulin in which Gln-3 and Thr-146 have We have begun to investigate the role of the central helix both been replacedby cysteines
Calmodulin was isolated from bovine brain as described by Gopalakrishna and Anderson (1982).Calmodulin expressed in E. coli was isolated as described by Craiget al. (1987).Protein concentrations were determined as described by Bradford (1976),using bovine serum albumin and bovine calmodulin standards. [Q3C,T146C]calmodulin’ was converted to thereduced form by incubation with 5 mM DTT at 30 “Cfor 1h, followed by addition of potassium acetateto a final concentration of 40 mM
During dialysis against 10 mM ammonium This class of model is consistent with small angle x-ray bicarbonate, which is the final step in purification, this cys- scattering investigations of calmodulin in solution, which teine pair is oxidized to an intramolecular disulfide bridge. indicate that the average conformation is extended, This is indicated by the marked reduction in SDS-gelelectro- as seen in the crystal structure, there is probably segmental phoretic mobility seen after incubatingpurified [Q3C, T146Cl flexibility within the central helix (Heidorn and Trewhella, calmodulin with dithiothreitol (Fig. 1).The mobility of re- 1988; Seaton et aL, 1985)
Summary
Altered calmodulin in which Gln-3 and Thr-146 have We have begun to investigate the role of the central helix both been replacedby cysteines. Mixed rabbit skeletal muscle activation of myosin light chain kinase activity by the cross-linkedand cross-linked/trypsinizedproteins are not significantly different from those for the control protein.Thisresultindicatesthatoneroleforthe central helix may be to serve as a flexible tether between the calmodulin lobes.
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