Biomaterial Database

Denaturant unfolding of the ferric enterobactin receptor and ligand-induced stabilization studied by site-directed spin labeling. 1995

C S Klug, and W Su, and J Liu, and P E Klebba, and J B Feix

Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.

FepA is an integral outer membrane protein that is the specific receptor for the siderophore, ferric enterobactin, and is thus primarily responsible for iron uptake in many Gram-negative bacteria. A site-specific mutant of FepA, containing a single introduced cysteine in the ligand-binding domain, was spin labeled and used to examine the denaturant-induced unfolding of this receptor with guanidine hydrochloride (Gdn-HCl) and urea. Electron spin resonance (ESR) spectra showed conversion of the spin label from a motionally-restricted, immobilized environment to a freely-accessible, rotationally-mobile state upon denaturation. Unfolding was also followed by nondenaturing polyacrylamide gel electrophoresis (PAGE), which is sensitive to loss of the putative transmembrane beta-structure, and displayed a similar concentration dependence. Unfolding occurred over relatively narrow ranges of denaturant concentration, indicating a high degree of cooperativity. Unfolding was fully reversible under the conditions employed. Rapid, spontaneous refolding occurred in the presence of Triton X-100 and did not require exogenous lipids. Refolding could be induced by either dialysis, dilution to low denaturant concentration, or ethanol precipitation. At ambient temperature the free energy of unfolding extrapolated to zero denaturant concentration (delta GU zero) was 6.24 +/- 0.63 kcal/mol. Values of delta GU zero obtained with Gdn-HCl and urea were in good agreement, as were values obtained from linear extrapolation and nonlinear regression fitting to a two-state equilibrium. This is the first report of a quantitative evaluation of the free energy of unfolding for an integral membrane protein.

UI	MeSH Term	Description	Entries
D007501	Iron	A metallic element with atomic symbol Fe, atomic number 26, and atomic weight 55.85. It is an essential constituent of HEMOGLOBINS; CYTOCHROMES; and IRON-BINDING PROTEINS. It plays a role in cellular redox reactions and in the transport of OXYGEN.	Iron-56,Iron 56
D008024	Ligands	A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)	Ligand
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011489	Protein Denaturation	Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein.	Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D011956	Receptors, Cell Surface	Cell surface proteins that bind signalling molecules external to the cell with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell (From Alberts, Molecular Biology of the Cell, 2nd ed, pp693-5). Cell surface receptors, unlike enzymes, do not chemically alter their ligands.	Cell Surface Receptor,Cell Surface Receptors,Hormone Receptors, Cell Surface,Receptors, Endogenous Substances,Cell Surface Hormone Receptors,Endogenous Substances Receptors,Receptor, Cell Surface,Surface Receptor, Cell
D002352	Carrier Proteins	Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes.	Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D004578	Electron Spin Resonance Spectroscopy	A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING.	ENDOR,Electron Nuclear Double Resonance,Electron Paramagnetic Resonance,Paramagnetic Resonance,Electron Spin Resonance,Paramagnetic Resonance, Electron,Resonance, Electron Paramagnetic,Resonance, Electron Spin,Resonance, Paramagnetic
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004758	Enterobactin	An iron-binding cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine. It is produced by E COLI and other enteric bacteria.	Enterochelin
D001425	Bacterial Outer Membrane Proteins	Proteins isolated from the outer membrane of Gram-negative bacteria.	OMP Proteins,Outer Membrane Proteins, Bacterial,Outer Membrane Lipoproteins, Bacterial