Combining EPR with fluorescence spectroscopy to monitor conformational changes at the myosin nucleotide pocket.

Nariman Naber, A. Málnási-Csizmadia, Thomas J. Purcell, Roger Cooke, Edward Pate

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WTSLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin myosin diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins. (c) 2009 Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)937-948
Number of pages12
JournalJournal of Molecular Biology
Volume396
Issue number4
DOIs
Publication statusPublished - Mar 5 2010

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Fluorescence Spectrometry
Electron Spin Resonance Spectroscopy
Myosins
Nucleotides
Actins
Tryptophan
Diphosphates
Actomyosin
Dictyostelium
Adenosine Diphosphate
Spectrum Analysis
Proteins
Binding Sites
Muscles

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Combining EPR with fluorescence spectroscopy to monitor conformational changes at the myosin nucleotide pocket. / Naber, Nariman; Málnási-Csizmadia, A.; Purcell, Thomas J.; Cooke, Roger; Pate, Edward.

In: Journal of Molecular Biology, Vol. 396, No. 4, 05.03.2010, p. 937-948.

Research output: Contribution to journalArticle

Naber, Nariman ; Málnási-Csizmadia, A. ; Purcell, Thomas J. ; Cooke, Roger ; Pate, Edward. / Combining EPR with fluorescence spectroscopy to monitor conformational changes at the myosin nucleotide pocket. In: Journal of Molecular Biology. 2010 ; Vol. 396, No. 4. pp. 937-948.
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