We observed specific NDE1 NDEL1 interaction and NDEL1 self-association, although their binding was significantly weaker than the noted DISC1 NDEL1 interaction (Fig. 5A).
Analyzed by western-blotting and densitometry, Gαo1 strongly bound RGS4 in the presence of GMP-PNP, whereas GDP- and GTP-preloaded Gαo1/RGS4-binding was significantly weaker (Fig 3b).
Since ATP (and ADP) binding to SNF1 is significantly weaker than for the coumarin adducts, it is not possible to establish a robust estimate of the binding constant for Mg.ATP binding at the weaker site.
The titration also shows that the binding of L6RM to G3P is significantly weaker than in wild-type cTIM.
Consistent with earlier observations showing that the conformation of mammalian Merlin was dynamic in its native state, the binding of Merlin-CTD to Merlin-FERM is significantly weaker (Kd of ∼3 μM, Supplementary information, Figure S1A) than the corresponding interaction between Moesin's FERM domain and its tail domain (Kd of ∼0.016 μM, Supplementary information, Figure S1B).
Unexpectedly, binding of NEMO to K63-linked di-ubiquitin is significantly weaker than to linear chains [ 17, 18, 26], suggesting that linear polyubiquitin chains may be the true physiological targets [ 18].
Affinity for this second site is significantly weaker, and thus it is possible that binding of the first molecule of di-ubiquitin may occlude binding of a second.
Yet the binding strength of CGB to the IP3Rs at a near physiological pH 7.5 is significantly weaker than that at the intragranular pH 5.5 [85], [85], and as a result the IP3R/Ca2+ channel-activating effect of CGB at this pH is markedly weaker than that shown at pH 5.5 [78], [78].
However, using an approach unbiased to antibody sensitivity, we compared binding affinities and found that SAP130 interacts strongly with Cul1 whereas the interaction with Skp1 and Skp2 is significantly weaker or undetectable, respectively.
