EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine

William Huang, Gregory J. Wilson*, Louise J. Brown, Harold Lam, Brett D. Hambly

*Corresponding author for this work

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 ≃14-50 μM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. and 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, τ(R) (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 (≃10 ns). TFP progressively recruited the probes into populations with τ(R) sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of ≃370-800 μM. 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.

Original languageEnglish
Pages (from-to)457-465
Number of pages9
JournalEuropean Journal of Biochemistry
Volume257
Issue number2
DOIs
Publication statusPublished - 15 Oct 1998
Externally publishedYes

Keywords

  • Calcium-binding proteins
  • Drug binding
  • Myosin light chains
  • Protein structure
  • Trifluoperazine

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