Table 1.

Embedded and Peripheral Lipid Interactions Enhance EnvZ His²⁴³ Autophosphorylation

	ADP Produced (Mole/Min per Mole of Enzyme)
	Low Osmolality	High Osmolality
EnvZc	0.09 ± 0.04	0.32 ± 0.08
EnvZ-E. coli nanodiscs	1.23 ± 0.17	4.9 ± 0.01
EnvZ-DOPC nanodiscs	0.93 ± 0.06	1.36 ± 0.05
EnvZc + nonembedded E. coli lipid nanodiscs	2.2 ± 0.33	4.4 ± 0.4
EnvZ-DDM	2.5 ± 0.1	7.3 ± 0.27

EnvZ His²⁴³ autophosphorylation in different membrane environments was measured using an in vitro ADP Glo kinase assay, which quantitates the amount of ADP produced in a kinase reaction via a luminescence-based detection method. These measurements were performed under conditions of both low (100 mOsm/kg) and high (20% sucrose/780 mOsm/kg) osmolality. Full-length EnvZ exhibited higher basal activity relative to EnvZc in E. coli lipid and DOPC nanodiscs, as well as DDM micelles. Increased autophosphorylation was also observed in EnvZc in the presence of nonembedded E. coli lipid nanodiscs. A ∼4-fold increase in kinase autophosphorylation was observed with increased osmolality in all environments. Activity is expressed in mol ADP/mol EnvZ per min.