Our findings that F NMR underestimated the true cooperativities of MZ1-induced ternary complexes suggested that similar issue would be faced when attempting to study hetero-bifunctional PROTACs with binary Kd at VHL that are significantly lower than the concentration of VHL required for performing the experiment. We hypothesized that the assay could offer much more accurate estimates of α in cases where the PROTAC has weaker VHL binding. We therefore performed measurements with a fluorinated analogue of MZ1 (15b, Figure ) and spy molecule 19. PROTAC 15b, like MZ1, is a Brd4selective degrader that forms a highly cooperative ternary complex in the presence of Brd4(2) (α = 15). However, 15b exhibited ~10-fold weaker binary VHL binding affinity compared to MZ1 (Kd VBC = 600nM; 24 c.f. 66nM for MZ1 ) due to unfavourable effects of the added fluorine atom in this particular stereochemistry. As 15b possesses a weaker affinity to VBC, the IC50 obtained in the F NMR titration experiments were farther from the tight binding limit met with MZ1. Consequently, a clear distinction upon the addition of Brd4(2) could be observed (Figure ) and the α value correlated more closely with that previously obtained by ITC (Table ). While the vast majority of PROTACs are hetero-bifunctional, i.e. the two binding ligands recruit different proteins, we have shown proof-of-concept for homo-bifunctional PROTACs that dimerize the VHL E3 ligase as a strategy to induce E3 ligase selfdegradation, i.e. homo-PROTACs. The same homo-PROTAC concept was later applied with the E3 ligase CRBN. In the same manner as hetero-bifunctional PROTACs, homo-PROTACs can form cooperative 2:1 complexes if the binding affinity to form the 2:1 complex is greater than that of the single warhead ligand to form a 1:1 complex. We therefore asked how our F NMR assay would perform with such systems in which the homo-PROTAC molecule dimerizes a monomeric E3 ligase. To address this, we performed measurements with spy molecule 19 and CM11, a VHL homo-PROTAC bearing two instances of VHL ligand, that induces the self-degradation of VHL by simultaneously binding to two molecules of the E3 ligase (Figure ). To provide suitable negative controls for VHL binding, we included compounds (Figure ) whereby the transhydroxyproline of the VHL ligand is replaced with the cis-epimer that instead abrogates VHL binding: CMP98 (trans-cis) which can only engage one VHL complex at a time, and the nonbinding control CMP99 (cis-cis). Pleasingly, the expected binding mechanisms for each of CM11 and its negative control compounds were reflected in the displacement profiles of spy molecule 19 (Figure ). CM11 was found to fully displace the spy molecule at low concentrations due to its 2-to-1 binding mode and high cooperativity, exhibiting a significant left-ward shift compared to CMP99 which can only undergo a 1-to-1 mode. The cooperativity value measured by F NMR showed good correlation with the ITC values previously reported (Table ). As expected, the non-binding control CMP98 did not displace the spy molecule (Figure ). Together these results highlight the applicability of F NMR competition experiments to measure cooperativity of both heterobifunctional PROTACs with weak binding affinity for one of the partner proteins, as well as for homo-PROTAC dimerizers.