Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.
J Phys Chem Lett
; 15(40): 10252-10257, 2024 Oct 10.
Article
in En
| MEDLINE
| ID: mdl-39360979
ABSTRACT
Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (â¼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Peptides
/
Protein Binding
/
Src Homology Domains
/
Fluorescence Polarization
/
Fluorescent Dyes
Language:
En
Journal:
J Phys Chem Lett
/
J. phys. chem. lett
/
The journal of physical chemistry letters
Year:
2024
Document type:
Article
Affiliation country:
Italy
Country of publication:
United States