Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands.
Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle(12)]-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle(12)]-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle(12)]-alpha-factor or [Orn7(NBD),Nle(12)]-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.
Ding
F X
FX
Department of Chemistry, The College of Staten Island of the City University of New York, Staten Island, New York 10314, USA.
Lee
B K
BK
Hauser
M
M
Davenport
L
L
Becker
J M
JM
Naider
F
F
D009327:4-Chloro-7-nitrobenzofurazan / Q000138:chemical synthesis / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D008024:Ligands; D000072235:Mating Factor; D010455:Peptides / Q000378:metabolism*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008024:Ligands; D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D009327:4-Chloro-7-nitrobenzofurazan