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.

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.

Detection and localization of occult lesions using breast magnetic resonance imaging: initial experience in a community hospital.

RATIONALE AND OBJECTIVES: To evaluate the outcome of diagnostic breast MR imaging followed by MR guided needle localization for mammographically and sonographically occult breast lesions in a community-based hospital. MATERIALS AND METHODS: Records of the initial 50 consecutive patients who underwent MR guided needle localizations at our institution from November 2001 to January 2003 were reviewed. Sixty-two lesions were localized by MR and were mammographically and sonographically occult. Pathology following excision was reviewed and correlated with the MR findings. RESULTS: Cancer was present in 15 % (9/62) of lesions or 18 % (9/50) of the women localized. Five of the lesions (56%) were invasive carcinoma and four (44%) were ductal carcinoma in situ (DCIS). High-risk lesions, including atypical ductal hyperplasia (ADH) and atypical lobular hyperplasia (ALH), were found in 6.5 % (4/62) of the lesions, while 3 % (2/62) of the lesions contained lobular carcinoma in situ (LCIS). Cancer plus high risk lesions were found in 15/62 (24%) lesions or 14/50 (28%) of women who underwent biopsy. CONCLUSION: The data in this study supports findings from other studies conducted by large research institutions. In this regard, it is important that community-based hospitals, such as the one operating this breast MR program, can achieve the same positive predictive values as those found in data emanating from academic institutions.

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae.

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.

Synthetic peptides as probes for conformational preferences of domains of membrane receptors.

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 alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues.