期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
卷 1808, 期 11, 页码 2674-2684出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2011.07.011
关键词
GPCR; Membrane protein structure; Fragment; Transmembrane
资金
- National Institutes of Health [GM22086, GM22087]
- Volkswagen Foundation
- Deutsche Forschungsgemeinschaft (DFG)
- Japan Society for the Promotion of Science (JSPS)
- New York State Office of Science, Technology, and Academic Research
- National Institutes of Health, USA
- Keck Foundation
- New York City Economic Development Corp.
- New York State
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 alpha-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. (C) 2011 Elsevier B.V. All rights reserved.
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