title: Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer.
abstract: Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a (2) H-labeled d8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy.
authors.affiliation: Cell, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, Ohio, 45056.|Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Rongfu
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Kaylee R
authors.initials: KR
authors.lastname: Gibson
authors.affiliation: Cell, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, Ohio, 45056.|Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Nefertiti B
authors.initials: NB
authors.lastname: Muhammad
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Avnika P
authors.initials: AP
authors.lastname: Bali
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Raven G
authors.initials: RG
authors.lastname: Comer
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Lishan
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Craig
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Mccarrick
authors.affiliation: Cell, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, Ohio, 45056.|Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Carole
authors.initials: C
authors.lastname: Dabney-Smith
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, 37232.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Sanders
authors.affiliation: Cell, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, Ohio, 45056.|Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, 45056.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D024642:Potassium Channels, Voltage-Gated; D017433:Protein Structure, Secondary
chemical_list: C489622:KCNE1 protein, human; D008051:Lipid Bilayers; D008565:Membrane Proteins; D024642:Potassium Channels, Voltage-Gated
keywords: EPR; ESEEM; KCNE1; SDSL; membrane protein; α-helix