| Title: | Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor |
| Author(s): | Estephan R; Englander J; Arshava B; Samples KL; Becker JM; Naider F; |
| Address: | "Department of Chemistry, College of Staten Island and Macromolecular Assemblies Institute of the City University of New York, Staten Island, New York 10314, USA" |
| ISSN/ISBN: | 0006-2960 (Print) 0006-2960 (Linking) |
| Abstract: | "The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis" |
| Keywords: | "Amino Acid Sequence Chromatography, High Pressure Liquid Cyanogen Bromide Mass Spectrometry Molecular Sequence Data Nitrogen Isotopes Nuclear Magnetic Resonance, Biomolecular Protein Conformation Protein Structure, Secondary Protein Structure, Tertiary Re;" |
| Notes: | "MedlineEstephan, Racha Englander, Jacqueline Arshava, Boris Samples, Karen L Becker, Jeffrey M Naider, Fred eng GM22086/GM/NIGMS NIH HHS/ GM22086-27S1/GM/NIGMS NIH HHS/ GM22087/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. 2005/09/01 Biochemistry. 2005 Sep 6; 44(35):11795-810. doi: 10.1021/bi0507231" |
