Analysis of ligand-receptor cross-linked fragments by mass spectrometry

Son, Çağdaş Devrim
Hurst, GB
Naider, F
Becker, JM
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.


Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver
İşgör, Sultan Belgin; Çoruh, Nursen; Department of Biochemistry (2004)
The glutathione-S-transferases (GSTs) (EC. are enzymes that participate in cellular detoxification of endogenous as well as foreign electrophilic compounds, function in the cellular detoxification systems and are evolved to protect cells against reactive oxygen metabolites by conjugating the reactive molecules to the nucleophile scavenging tripeptide glutathione (GSH, ?-glu-cys-gly). The GSTs are found in all eukaryotes and prokaryotic systems, in the cytoplasm, on the microsomes, and in the mitoch...
Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.
Celić, A; Martin, NP; Son, Çağdaş Devrim; Becker, JM; Naider, F; Dumont, ME (American Chemical Society (ACS), 2003-03-18)
The α-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228−Leu248) to intensive random mutagenesis and screened multiply substituted alleles...
Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment.
Huang, LY; Umanah, G; Hauser, M; Son, Çağdaş Devrim; Arshava, B; Naider, F; Becker, JM (American Chemical Society (ACS), 2008-05-20)
Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone cc factor of Saccharomyces cerevisiae. This receptor- pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl-L-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid ...
Investigation for natural extract inhibitors of bovine lens aldose reductase responsible for the formation of diabetis dependent cataract
Onay, Melih; Çoruh, Nursen; Department of Biochemistry (2008)
In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dep...
KOCABIVIK, S; PERLIN, MH (Elsevier BV, 1994-01-01)
1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme. 2. The mutations of Gly205 --> GIu, Gly210 --> Ala and Arg211 --> Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418. 3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg2+ ATP. 4. Alternatively substitutions at a highly c...
Citation Formats
Ç. D. Son, G. Hurst, F. Naider, and J. Becker, “Analysis of ligand-receptor cross-linked fragments by mass spectrometry,” JOURNAL OF PEPTIDE RESEARCH, pp. 418–426, 2005, Accessed: 00, 2020. [Online]. Available: