title: Spin-echo EPR of Na,K-ATPase unfolding by urea.
abstract: Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II -SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.
authors.affiliation: Dipartimento di Fisica and UdR CNISM, Università della Calabria, 87036 Arcavacata di Rende (CS), Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Guzzi
authors.forename: Mohammad
authors.initials: M
authors.lastname: Babavali
authors.forename: Rosa
authors.initials: R
authors.lastname: Bartucci
authors.forename: Luigi
authors.initials: L
authors.lastname: Sportelli
authors.forename: Mikael
authors.initials: M
authors.lastname: Esmann
authors.forename: Derek
authors.initials: D
authors.lastname: Marsh
mesh_terms: D000465:Algorithms; D000818:Animals; D041961:Deuterium Exchange Measurement; D017666:Deuterium Oxide / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D058767:Protein Unfolding / Q000187:drug effects*; D012491:Salt Gland / Q000201:enzymology; D012754:Sharks; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D014508:Urea / Q000494:pharmacology*; D014867:Water / Q000737:chemistry
chemical_list: D029941:Fish Proteins; D021122:Protein Subunits; D013113:Spin Labels; D014867:Water; D014508:Urea; D000254:Sodium-Potassium-Exchanging ATPase; D017666:Deuterium Oxide