EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.
The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.
Huang
W
W
Department of Pathology, University of Sydney, NSW, Australia.
Wilson
G J
GJ
Brown
L J
LJ
Lam
H
H
Hambly
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D014150:Antipsychotic Agents / Q000378:metabolism*; D002412:Cations; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014268:Trifluoperazine / Q000378:metabolism*
D014150:Antipsychotic Agents; D002412:Cations; D018994:Myosin Light Chains; D014268:Trifluoperazine