The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin.
The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.
Garone
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Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Albaugh
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Steiner
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RF
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D004789:Enzyme Activation; D005895:Gizzard, Avian / Q000201:enzymology; D008297:Male; D008956:Models, Chemical; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014422:Turkeys
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase