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Reference: Cohen LS, et al. (2011) Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments. Biochim Biophys Acta 1808(11):2674-84

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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 a-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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't | Research Support, N.I.H., Extramural
Authors
Cohen LS, Arshava B, Neumoin A, Becker JM, Guntert P, Zerbe O, Naider F
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