Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins.
The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-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 rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-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 rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta 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 rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.
Novy
R E
RE
Department of Biological Sciences, University of Iowa, Iowa City 52242.
Liu
L F
LF
Lin
C S
CS
Helfman
D M
DM
Lin
J J
JJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002645:Chickens; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D011189:Potassium Chloride; D004247:DNA; D008274:Magnesium