Hook effect characterization
of a highly cooperative ternary complex
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
0.92
0.96
1.00
1.04
1.08
1.12
Ratio
670nm/
650nm
MZ1 (nM)
Binary complex BRD2 + MZ1
Ternary complex BRD2 + MZ1 + VCB
The binary and ternary complex formed by target BRD2, PROTAC MZ1,
and ligase VCB were characterized with measurements performed in solution
with labeled BRD2 using Dianthus with Spectral Shi . To calculate the affinity
constants the data obtained were fit using a model for three-component systems
1
.
The affinity of the binary complex BRD2 + MZ1 was determined first (K
d
= 18.8 nM),
followed by the characterization of the ternary complex BRD2 + MZ1 + VCB
under conditions that revealed the hook effect. The concentration of MZ1 that results
in maximum ternary complex formation was determined to be 57 nM.
Additionally, the formation of the ternary complex confirmed previously reported
positive cooperativity
2
(a = 9.3) which is known to correlate with efficient
ubiquitination and target degradation.
Dianthus with Spectral Shi offers in-solution measurements and options to label
the POI or ligase, plus generates data in good agreement with a model for
three-component binding equilibria for results you feel confident about.
1
Douglass et al J Am Chem Soc (2013), 135, 6092-6099
2
Zengerle et al ACS Chem Biol (2015), 10, 1770-1777
