Journal of Peptide Science J. Pept. Sci. 2006; 12: 808–822 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.816 Effects of N- and C-terminal addition of oligolysines or native loop residues on the biophysical properties of transmembrane domain peptides from a G-protein coupled receptor ‡ PATRICIA CANO-SANCHEZ, a BEATRICE SEVERINO, a V. V. SURESHBABU, a JOE RUSSO, a TATSUYA INUI, a FA-XIANG DING, a BORIS ARSHAVA, a JEFF BECKER b and FRED NAIDER a * a Department of Chemistry, College of Staten Island and Macromolecular Assemblies Institute of the City University of New York, Staten Island, New York 10314, USA b Department of Microbiology and Graduate Program in Genome Science and Technology, University of Tennessee, Knoxville, Tennessee 37996, USA Received 20 April 2006; Revised 9 May 2006; Accepted 10 May 2006 Abstract: Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N - and C-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lys n (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol–water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies. Copyright  2006 European Peptide Society and John Wiley & Sons, Ltd. Keywords: transmembrane peptides; circular dichroism; oligolysines; peptide structure INTRODUCTION The G-protein coupled receptor (GPCR) super family includes several thousand distinct but related pro- teins. They are found in a wide range of organisms and are involved in the transmission of signals across membranes [1]. Although the receptors are conserved in structure, the ligands span a large range of vastly diverse entities from peptides, small molecules to pro- teins [2]. They are composed of a single polypeptide chain containing seven regions of 20–28 hydrophobic amino acids that represent transmembrane domains * Correspondence to: F. Naider, Leonard and Esther Kurtz Term Professor, Department of Chemistry, College of Staten Island of The City University of New York, 2800 Victory Blvd, Staten Island, New York 10314, USA; e-mail: naider@mail.csi.cuny.edu ‡ Dedication: Professor Murray Goodman initiated a multidisciplinary approach to study the biophysical properties of oligopeptides. His analyses contained careful design of model compounds, efficient synthetic routes and detailed biophysical analyses under a variety of conditions. His laboratory was the first to use fluoroalcohols in the conformational analysis of peptides paving the way for literally thousands of investigations using these versatile solvents, which are now considered to represent a membrane mimetic environment. The studies presented herein on peptides corresponding to transmembrane regions of a G-protein coupled receptor follow the ‘Goodman method’ and we will always remain indebted to our Professor and Teacher for his guidance and inspiration. (TMDs) [3]. The TM segments form α-helices, oriented roughly perpendicular to the membrane as shown in rhodopsin [4]. Structural analysis by techniques such as nuclear magnetic resonance (NMR) or X-ray crystal- lography of the TMDs of integral membrane proteins has traditionally been impeded by their hydrophobic nature and their tendency to form intractable aggre- gates in the presence of even relatively small amounts of water. This behavior is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and likely occurs on high performance liquid chro- matography (HPLC) columns during peptide purifica- tion. Obtaining large amounts of TMD samples exper- imentally through biosynthesis or chemical synthesis is complicated by the fact that these polypeptides are inherently hydrophobic and often require nonstandard solubilization and purification strategies [5,6]. A number of studies have used charged/polar residues at the termini of hydrophobic peptides to prevent unwanted nonspecific aggregation of peptides [7–12]. Model TM peptides flanked with Lys residues have already been studied to investigate how TM peptides and bilayers influence each other’s biophysical properties [13–16]. It has been reported that Lys residues added to the end of single-spanning TMDs Copyright  2006 European Peptide Society and John Wiley & Sons, Ltd.