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Fred Naider, Boris Arshava, Haibo Xie, Shi-feng Liu, Woei Y. Eng, Shu-Hua Wang, Kathleen Valentine, Gianluigi Veglia, Francesca Marassi, Stanley J. Opella, and Jeffrey M. Becker 1 College of Staten Island, CUNY, S.I., NY 10314, U.S.A.; 2 University of Pennsylvania, Philadelphia, PA 19104, U.S.A.; Wistar Institute, Philadelphia, PA 19104, U.S.A.; and University of Tennessee, Knoxville, TN 37996, U.S.A.

High-resolution NMR spectroscopy of liquids has become a standard method for conformational studies in biochemistry. This method provides information on the secondary and tertiary structure of macromolecules and on intermolecular interactions such as ligand receptor binding. These determinations are based mainly on interatomic distance measurements by quantitative analysis of NOESY spectra and, to a lesser degree, on torsion angle measurements based on homoor heteronuclear scalar coupling constants. As with liquids, internuclear distances in solids are reflected in dipole-dipole coupling constants between the nuclear magnetic dipoles. Rotational-echo double resonance (REDOR) spectroscopy has been employed to determine heteronuclear dipole-dipole coupling constants [1,2] and to study internuclear distances in peptides, proteins and bound ligands. An important target for the REDOR experiment is an amorphous solid. For biologically important amorphous solids that cannot be obtained in crystalline form, NMR spectroscopy is the only method for direct investigation of conformation at the atomic level. In the present study we examine the REDOR of selectively labeled peptide hormone in lyophilized powders. In order to understand the contribution of naturalabundance nuclei to the REDOR data at long evolution times, we have used a three-body calculation [3,4] and assumed a spherical distance distribution for natural abundance corrections. In addition we have evaluated the REDOR results in terms of a superposition of different distances with appropriate weighting factors. These approaches allow us to fit the experimental data on the to a high degree of accuracy and to conclude that this peptide has a tendency to be bent in a lyophilized powder.

Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s. A pioneer in this field was Dr. Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides. This article reviews some of Dr. Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae. Peptides containing 30–40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography. CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles. Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the α-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains. NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule. The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies.

A detailed analysis of the structure of an 18-residue peptide AQSLLVPSIIFILAYSLK [M6(252–269, C252A)] in 1,2-dimyristoyl-sn-glycero-phosphocholine bilayers was carried out using solid state NMR and attenuated total reflection Fourier transform infrared spectroscopy. The peptide corresponds to a portion of the 6th transmembrane domain of the α-factor receptor of Saccharomyces cerevisiae. Ten homologs of M6(252–269, C252A) were synthesized in which individual residues were labeled with 15N. One- and two-dimensional solid state NMR experiments were used to determine the chemical shifts and 1H–15N dipolar coupling constants for the 15N-labeled peptides in oriented dimyristoylphosphatidylcholine bilayers on stacked glass plates. These parameters were used to calculate the structure and orientation of M6(252–269, C252A) in the bilayers. The results indicate that the carboxyl terminal residues (9–14) are α-helical and oriented with an angle of about 8° with respect to the bilayer normal. Independently, an attenuated total reflection Fourier transform infrared spectroscopy analysis on M6(252–269, C252A) in a 1,2-dimyristoyl-sn-glycero-phosphocholine bilayer concluded that the helix tilt angle was about 12.5°. The results on the structure of M6(252–269, C252A) in bilayers are in good agreement with the structure determined in trifluoroethanol/water solutions (B. Arshava et al. Biopolymers, 1998, Vol. 46, pp. 343–357). The present study shows that solid state NMR spectroscopy can provide high resolution information on the structure of transmembrane domains of a G protein-coupled receptor. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 243–256, 2001

Coral reef communities are an important part of the marine ecosystem. They are highly reproductive and apart from the tropical rain forest, there is no natural environment so rich in species diversity as the marine coral reef. During last two decades, there have been an increasing number of reports of disease of corals referred to as coral bleaching, which breaks the symbiotic association between the coral hosts and their photosynthetic micro algal endosymbionants known as zooxanthellae. In an attempt to reveal the actual mechanism behind coral bleaching earlier we documented that a pathogenic bacterium Vibrio shiloi was the casuative agent involved in the coral bleaching [1]. This coral pathogen (V. shiloi) produces an array of extracellular material that can inhibit photosynthesis, bleach and lyse zooxanthellae. Recently we isolated a proline-rich dodecapeptide (PYPVYAPPPVVP) from Vibrio shiloi named Toxin P and its structure and activity was confirmed by chemical synthesis [2]. The detail conformational analysis of synthetic Toxin P was carried out using CD and NMR in various media including membrane mimetic environments.

The yeast G protein coupled-receptor (Ste2p) is essential for the mating of MAT a cells with their counterpart MATα cells. Mutation of Proline 256 located in the 6th transmembrane domain of this receptor constitutively activates it in the absence of the α-factor pheromone that is the normal agonist [1]. This phenotype indicates that the single amino acid change in residue 256 may change the overall structure of this membrane protein. Based on this conclusion, attempts are being made to determine the structure of the domain of Ste2p comprising the 5th and 6th transmembrane domains joined by the third intracellular loop (M5I3M6), and to correlate its molecular arrangement with the genetic results mentioned above. In order to produce this domain we inserted it into an expression plasmid that fused M5I3M6 to an N-terminal leader protein called TrpALE. The resulting fusion protein can be overexpressed in E. coli, isolated, and purified using affinity chromatography and/or HPLC. The link between the TrpΔLE and M5I3M6 occurs at a methionine residue, allowing cleavage of the fusion junction with CNBr. Therefore, naturally occurring methionines located in the 5th and the 6th transmembranes were mutated to leucines or alanines. After characterization of the biological activity and ligand binding affinities of these mutant receptors, it was decided to study the receptor containing the double mutation Met218Leu, Met250Ala, since it exhibited characteristics most similar to those of wild-type Ste2p.

The α-factor tridecapeptide initiates mating in Saccharomyces cerevisiae upon interaction with Ste2p, its cognate G-protein coupled receptor (GPCR). This interaction is being used as a paradigm for understanding the structure and mechanism of activation of GPCRs by medium-sized peptides. In this article, the use of fragments of Ste2p to study its structure is reviewed. Methods of synthesis of peptides corresponding to both extramembranous and transmembrane domains of Ste2p are evaluated and problems that are encountered during synthesis and purification are described. The results from conformational analyses of the peptide fragments using fluorescence spectroscopy, CD, infrared spectroscopy, and NMR spectroscopy in organic-aqueous mixtures and in the presence of detergent micelles and lipid bilayers are critically reviewed. The data obtained to date provide biophysical evidence for the structure of different domains of Ste2p and indicate that peptides corresponding to these domains have unique biophysical tendencies. The studies carried out on Ste2p fragments indicate that valuable information concerning the structure of the intact receptor can be obtained by studying peptide fragments corresponding to domains of these polytopic integral membrane proteins. © 2002 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 60: 334–350, 2001