Jeffrey M. Becker

Peptide transport in Candida albicans: implications for the development of antifungal agents.

The yeast Candida albicans typically exists in a symbiotic relationship with humans (68). In the event, however, that the host is immunologically weakened upon challenge by disease or medical therapy, C. albicans can develop into a virulent pathogen. For many years this microorganism has gained increasing prominence as an opportunistic pathogen in hospital-associated infections and in compromised patients. Recently, this yeast has been documented as an agent in the morbidity and mortality of patients suffering from acquired immune deficiency syndrome (AIDS)(18).

Synthesis and Biophysical Characterization of a Multidomain Peptide from a Saccharomyces cerevisiae G Protein-coupled Receptor

We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the α-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mm concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.

The chain length dependence of helix formation of the second transmembrane domain of a G protein-coupled receptor of Saccharomyces cerevisiae.

The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the α-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disordered, and all of the other peptides assumed partially α-helical secondary structures in 99% trifluoroethanol (TFE)/H2O. In 50% TFE/H2O, M2-30 assumed a β-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H2O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed α-helical structures, whereas the other peptides formed β-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed β-structures in this environment. Another homologous 30-residue peptide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were α-helical and oriented at angles of 12°, 13°, 36°, and 34°, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.