Expression of Smooth-Muscle and Nonmuscle Tropomyosins in Escherichia-Coli and Characterization of Bacterially Produced Tropomyosins

Novy, R. E., Liu, L. F., Lin, C. S., Helfman, D. M., Lin, J. J. C. (March 1993) Expression of Smooth-Muscle and Nonmuscle Tropomyosins in Escherichia-Coli and Characterization of Bacterially Produced Tropomyosins. Biochimica Et Biophysica Acta - Protein Structure and Molecular Enzymology, 1162 (3). pp. 255-265. ISSN 0006-3002

URL: http://www.ncbi.nlm.nih.gov/pubmed/8457589
DOI: 10.1016/0167-4838(93)90289-4

Abstract

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSMbeta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSMbeta) and a mutant (rCSMbeta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSMbeta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 . 10(6) and 2.78 . 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSMbeta) appeared to have a comparable actin-binding affinity to that of gel-purified CSMbeta homodimer (1.25 . 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 . 10(7) M-1). The amino-terminal deletion mutant rCSMbeta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSMbeta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSMbeta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSMbeta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSMbeta in the actin binding and the caldesmon enhancement.

Item Type: Paper
Uncontrolled Keywords: TROPOMYOSIN CALDESMON COOPERATIVITY N-TERMINAL-DELETED TROPOMYOSIN SMOOTH MUSCLE FIBROBLAST COMPLETE NUCLEOTIDE-SEQUENCE CHICKEN-EMBRYO FIBROBLASTS ACTIN-BINDING PROPERTIES RAT CULTURED-CELLS ALPHA-TROPOMYOSIN F-ACTIN BETA-TROPOMYOSIN MESSENGER-RNAS MONOCLONAL-ANTIBODIES MULTIPLE ISOFORMS
Subjects: organism description > bacteria > escherichia coli
organs, tissues, organelles, cell types and functions > tissues types and functions > smooth muscle
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > tropomyosin
CSHL Authors:
Communities: CSHL labs > Helfman lab
Depositing User: Matt Covey
Date: 26 March 1993
Date Deposited: 21 Apr 2016 18:53
Last Modified: 21 Apr 2016 18:53
URI: http://repository.cshl.edu/id/eprint/32486

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