Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release

C. M. Yengo, L. Chrin, A. S. Rovner, C. L. Berger

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

The helix - loop - helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R- sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546- MDE [emission peak (λ(max)) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k(q)) = 6.4 M-1·ns-1] and V413W-MDE [λ(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M-1·ns-1] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average λ(max) of 337 or 336 nm, respectively, and protection from dimethyl(2- hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp- 413 has a single conformation with an average λ(max) of 338 nm in the ADP- bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (λ(max) = 341-347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent- exposed conformation upon ADP release.

Original languageEnglish (US)
Pages (from-to)14515-14523
Number of pages9
JournalBiochemistry
Volume38
Issue number44
DOIs
StatePublished - Nov 2 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry

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