title: Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.
abstract: Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.
authors.affiliation: Department of Chemistry, The College of Staten Island, City University of New York, Staten Island, NY 10314, USA.
authors.forename: L S
authors.initials: LS
authors.lastname: Cohen
authors.forename: B
authors.initials: B
authors.lastname: Arshava
authors.forename: A
authors.initials: A
authors.lastname: Neumoin
authors.forename: J M
authors.initials: JM
authors.lastname: Becker
authors.forename: P
authors.initials: P
authors.lastname: Güntert
authors.forename: O
authors.initials: O
authors.lastname: Zerbe
authors.forename: F
authors.initials: F
authors.lastname: Naider
mesh_terms: D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008823:Micelles; D010455:Peptides / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D008823:Micelles; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D014867:Water; D014270:Trifluoroethanol