We describe the development and characterization of substance P labeled at Lys3 with fluorescein ([fluorescein Lys3]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys3]SP is an agonist at the human NK1 receptor, with an affinity for both the high-affinity and low-affinity binding states of the receptor approximately 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directly by monitoring either a decrease in fluorescence intensity or an increase in anisotropy of the [fluorescein Lys3]SP. Detection by anisotropy gave the larger signal and thus was used to characterize the interaction of [fluorescein Lys3]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The fluorescence was quenched by about 15% upon binding to the receptor. Bound [fluorescein Lys3]SP was displaced by unlabeled SP and by the quinuclidine antagonist L-703,606. As expected for an agonist, binding was also reduced by the addition of the nonhydrolyzable guanine nucleotide analog GppNHp. [fluorescein Lys3]SP should provide a useful structural and kinetic probe for the NK1 receptor.
The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication. Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium. For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared. The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site. HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM. Binding was saturable and consistent with a single class of noninteracting sites. The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication. Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM). E1 binding to Fl-E1E2BS was of very low affinity. Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM. This effect was not dependent upon ATP or magnesium ion. Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur. In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each. The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.