• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biophysjLink to Publisher's site
Biophys J. Oct 2002; 83(4): 1854–1866.
PMCID: PMC1302278

Molecular dynamics simulation of site-directed spin labeling: experimental validation in muscle fibers.

Abstract

We have developed a computational molecular dynamics technique to simulate the motions of spin labels bound to the regulatory domain of scallop myosin. These calculations were then directly compared with site-directed spin labeling experimental results obtained by preparing seven single-cysteine mutants of the smooth muscle (chicken gizzard) myosin regulatory light chain and performing electron paramagnetic resonance experiments on these spin-labeled regulatory light chains in functional scallop muscle fibers. We determined molecular dynamics simulation conditions necessary for obtaining a convergent orientational trajectory of the spin label, and from these trajectories we then calculated correlation times, orientational distributions, and order parameters. Simulated order parameters closely match those determined experimentally, validating our molecular dynamics modeling technique, and demonstrating our ability to predict preferred sites for labeling by computer simulation. In several cases, more than one rotational mode was observed within the 14-ns trajectory, suggesting that the spin label samples several local energy minima. This study uses molecular dynamics simulations of an experimental system to explore and enhance the site-directed spin labeling technique.

Full Text

The Full Text of this article is available as a PDF (597K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baker JE, Brust-Mascher I, Ramachandran S, LaConte LE, Thomas DD. A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2944–2949. [PMC free article] [PubMed]
  • Brust-Mascher I, LaConte LE, Baker JE, Thomas DD. Myosin light-chain domain rotates upon muscle activation but not ATP hydrolysis. Biochemistry. 1999 Sep 28;38(39):12607–12613. [PubMed]
  • Columbus L, Kálai T, Jekö J, Hideg K, Hubbell WL. Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure. Biochemistry. 2001 Apr 3;40(13):3828–3846. [PubMed]
  • Dauber-Osguthorpe P, Roberts VA, Osguthorpe DJ, Wolff J, Genest M, Hagler AT. Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 1988;4(1):31–47. [PubMed]
  • Houdusse A, Cohen C. Structure of the regulatory domain of scallop myosin at 2 A resolution: implications for regulation. Structure. 1996 Jan 15;4(1):21–32. [PubMed]
  • Hubbell WL, Gross A, Langen R, Lietzow MA. Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol. 1998 Oct;8(5):649–656. [PubMed]
  • Hubbell WL, Mchaourab HS, Altenbach C, Lietzow MA. Watching proteins move using site-directed spin labeling. Structure. 1996 Jul 15;4(7):779–783. [PubMed]
  • Langen R, Oh KJ, Cascio D, Hubbell WL. Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure. Biochemistry. 2000 Jul 25;39(29):8396–8405. [PubMed]
  • Maita T, Chen JI, Matsuda G. Amino-acid sequence of the 20 000-molecular-weight light chain of chicken gizzard-muscle myosin. Eur J Biochem. 1981 Jul;117(2):417–424. [PubMed]
  • Maita T, Konno K, Ojima T, Matsuda G. Amino acid sequences of the regulatory light chains of striated adductor muscle myosins from Ezo giant scallop and Akazara scallop. J Biochem. 1984 Jan;95(1):167–177. [PubMed]
  • Mendelson R, Morris EP. The structure of the acto-myosin subfragment 1 complex: results of searches using data from electron microscopy and x-ray crystallography. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8533–8538. [PMC free article] [PubMed]
  • Nyitrai M, Hild G, Lukács A, Bódis E, Somogyi B. Conformational distributions and proximity relationships in the rigor complex of actin and myosin subfragment-1. J Biol Chem. 2000 Jan 28;275(4):2404–2409. [PubMed]
  • Ostap EM, White HD, Thomas DD. Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis. Biochemistry. 1993 Jul 6;32(26):6712–6720. [PubMed]
  • Persechini A, Hartshorne DJ. Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin. Biochemistry. 1983 Jan 18;22(2):470–476. [PubMed]
  • Ramachandran S, Thomas DD. Rotational dynamics of the regulatory light chain in scallop muscle detected by time-resolved phosphorescence anisotropy. Biochemistry. 1999 Jul 13;38(28):9097–9104. [PubMed]
  • Roopnarine O, Szent-Györgyi AG, Thomas DD. Microsecond rotational dynamics of spin-labeled myosin regulatory light chain induced by relaxation and contraction of scallop muscle. Biochemistry. 1998 Oct 13;37(41):14428–14436. [PubMed]
  • Squier TC, Thomas DD. Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes. Biophys J. 1989 Oct;56(4):735–748. [PMC free article] [PubMed]
  • Steinhoff HJ, Hubbell WL. Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins. Biophys J. 1996 Oct;71(4):2201–2212. [PMC free article] [PubMed]
  • Thomas DD, Cooke R. Orientation of spin-labeled myosin heads in glycerinated muscle fibers. Biophys J. 1980 Dec;32(3):891–906. [PMC free article] [PubMed]
  • Volkmann N, Hanein D, Ouyang G, Trybus KM, DeRosier DJ, Lowey S. Evidence for cleft closure in actomyosin upon ADP release. Nat Struct Biol. 2000 Dec;7(12):1147–1155. [PubMed]
  • Xie X, Harrison DH, Schlichting I, Sweet RM, Kalabokis VN, Szent-Györgyi AG, Cohen C. Structure of the regulatory domain of scallop myosin at 2.8 A resolution. Nature. 1994 Mar 24;368(6469):306–312. [PubMed]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...