We have previously shown that the PROTAC MZ1 forms highly cooperative, stable, and long-lived ternary complexes with BET bromodomains, with preference for BD2s over BD1s, and in particular for Brd4 BD2 , and that these biophysical characteristics of the ternary complex underpin a high level of target ubiquitination and drive potent and fast degradation activity of MZ1 with Brd4. We thus wondered to what extent the improvements in cellular activity that we observed with our set of PROTACs might be contributed from more favorable ternary complex formation. To address this question, we biophysically characterized all compounds in our PROTAC series by measuring both binary binding to VHL and ternary complex formation between VHL, PROTAC, and both BD1 and BD2 bromodomains of Brd4, Brd3 and Brd2 (Figure , SI Table ). We measured cooperativity across the entire set of eight PROTACs vs six bromodomains (48 combinations). We used a competitive FP assay in which a fluorescently labelled HIF-1α peptide probe bound to VHL is displaced by titrating either PROTAC alone (for binary binding) or by titrating PROTACs preincubated with individual BET bromodomains (for ternary complex binding). This allows us to calculate the cooperativity (α) of ternary complex formation (α = Kd binary / Kd ternary , Figure ). Strikingly, in all amide-to-ester matched pairs, the esters were 2 to 3-fold weaker at binding to VHL, with the largest difference being between 21 (Kd = 81 nM) and 25 (Kd = 248 nM). As expected, due to the additional benzyl methyl group present in the VH032 ligand, PROTACs 22 and 26 showed the strongest binary binding to VHL (Kd = 34 nM and 63 nM, respectively). Additionally, all esters were also 2-to 7-fold weaker than their amide counterparts at binding VHL pre-bound to each individual BET bromodomain, as measured by their ternary Kd values (cf. Fig. ). One hypothesis for this decreased binding affinity is the formation of a new IMHB between the new HBA present in the ester group and a HBD of an amide in the VH032 ligand. This could cause the rigid and relatively bulky JQ1 ligand to sit on top of the VHL protein, potentially causing unfavorable clashes and requiring an energetic penalty to allow binding to VHL. This is somewhat evident when switching from a PEG linker in 25 to a more alkyl linker in and 27 which has negligible effects on VHL binding; however, shortening the linker in 28 gave a 1.75-fold increase in affinity. The shorter linker decreases flexibility for the molecule to fold and form new IMHBs. Recently, others have shown how VHL targeting PROTACs can fold and form IMHBs in different solutions to change their TPSA. Further structural studies are warranted to fully assess this phenomenon in the PROTACs presented here.