title: Characterizing solution surface loop conformational flexibility of the GM2 activator protein.
abstract: GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.
authors.affiliation: Department of Chemistry, University of Florida , P.O. Box 117200, Gainesville, Florida 32611-7200, United States.
authors.forename: Jeffery D
authors.initials: JD
authors.lastname: Carter
authors.forename: Jordan D
authors.initials: JD
authors.lastname: Mathias
authors.forename: Edwin F
authors.initials: EF
authors.lastname: Gomez
authors.forename: Yong
authors.initials: Y
authors.lastname: Ran
authors.forename: Fang
authors.initials: F
authors.lastname: Xu
authors.forename: Luis
authors.initials: L
authors.lastname: Galiano
authors.forename: Nguyen Q
authors.initials: NQ
authors.lastname: Tran
authors.forename: Peter W
authors.initials: PW
authors.lastname: D'Amore
authors.forename: Christine S
authors.initials: CS
authors.lastname: Wright
authors.forename: Dhruva K
authors.initials: DK
authors.lastname: Chakravorty
authors.forename: Gail E
authors.initials: GE
authors.lastname: Fanucci
mesh_terms: D003545:Cysteine / Q000737:chemistry; D004548:Elasticity; D004578:Electron Spin Resonance Spectroscopy; D049289:G(M2) Activator Protein / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D013113:Spin Labels; D013696:Temperature; D013816:Thermodynamics
chemical_list: D049289:G(M2) Activator Protein; D011994:Recombinant Proteins; D012996:Solutions; D013113:Spin Labels; D003545:Cysteine