RESUMO
The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.
Assuntos
Receptores Acoplados a Proteínas G/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Conjugação Genética , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A simple and concomitant esterification method for the synthesis of methyl, ethyl, t-butyl, benzyl, and 9-fluorenylmethyl esters of Fmoc-/Boc-/Z-beta-homoamino acids employing Fmoc-/Boc-/Z-alpha-aminodiazoketones by Wolff rearrangement is described. The method offers good yield with purity.
Assuntos
Aminoácidos/síntese química , Ésteres/síntese química , Cetonas/química , Alquilação , Aminoácidos/química , Ésteres/químicaRESUMO
A new and efficient method for the synthesis of N(alpha)-Fmoc-/Boc-/Z-beta-amino acids using the two-step Arndt-Eistert approach is described. Fmoc-/Boc-/Z-alpha-Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc-/Boc-/Z-alpha-aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding beta-amino acids using PhCOOAg/dioxane/H2O.
Assuntos
Aminoácidos/síntese química , Fluorenos/síntese química , Acilação , Aminoácidos/química , Diazometano/química , Fluorenos/química , Flúor/química , EstereoisomerismoRESUMO
A range of N(alpha)-Fmoc-protected amino acids, including those that contain t-butyl moiety, have been synthesized by employing Fmoc-Cl utilizing the activated, commercial zinc dust-promoted synthesis of carbamates under neutral conditions. A general procedure is described that circumvents the oligomerization side reaction normally noticed in Schotten-Baumann conditions. It is a simple, convenient and clean method. Thus, Fmoc-amino acids are obtained in high yield (85-92%) and purity as checked by thin-layer chromatography, high-performance liquid chromatography and other physical methods.