.png){kind=icon}
.png){kind=icon}
Return
A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions
.png){kind=icon}
PRE
AI
DOI：10.1002/anie.201605964.png)
.png){kind=icon}
Original request for help.png){kind=icon}
Share.png){kind=icon}
Save
Abstract
En 中文
We have developed Forster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10(-9)-10(-5) M) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.
Keywords:
fluorescence
FRET
kinetics
protein engineering
protein-protein interactions
AI Summary
Key information extracted from the uploaded paper, including a brief overview, abstract, background, key highlights, visual analysis, and future outlook.
Journal
IF:
16.9
Papers:
5.6W
Citations:
53.0W