Hydrogen Exchange (high affinity binding) Patterson et al(1990)Science 249755-759 ncubate inD hr to days HN ChetehgRanneastyangeaerncaiaionntepmesenek小adahenceK all residues-there should be a sharp distinction between protected and Assumes that there are no co-operative unfolding events as a result of binding the other molecule L64 …free form bound form Protection factors (k/ 8湖 598 02 Protection factors3 30 nge time (h ours) Hydrogen Exchange (high affinity binding) Patterson et al (1990) Science 249 755-759 Incubate in D2O 1hr to days HN HN HN HN O DN HN DN/HN HN O Dissociate at low pH (H/D exchange is slow) and isolate labelled protein DN HN DN/HN HN O HSQC to identify remaining NHs Calculate the rate of intensity change after incubation in the presence (kbound) and absence (kfree) of the binding partner Calculate kfree/kbound for all residues - there should be a sharp distinction between protected and unprotected residues Assumes that there are no co-operative unfolding events as a result of binding the other molecule Exchange time (hours) free form bound form Protection factors (kfree/kbound): K60 >60 L64 >50 E66 8 D93 ~0 Residues divided into 2 groups: Protection factor ! 7 Protection factor " 3