His-tag beta-Galactosidase in heterogenous system

MOTTOLA M; PERILLO MA; FLORES, S; SANCHEZ JM

Cuidad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de las Sociedades de BioCiencias; 2017

SAIC, SAIB, SAI, SAA, SAB, SAFE, SAFIS, SAH, SAP

The Escherichiacoli β-galactosidase (β-Gal) is a widely used enzyme for basic andapplied research. Usually, the enzyme activity is measured in heterogeneousmedia. In our laboratory, we demonstrated that β-Gal can interact with lipid/water interfaces. In this condition weobserved an enhanced catalytic activity, an increase in thermostability and theprevention of β-Galproteolysis. Currently we are producing a recombinant β-GalHis ( 6 histidine residues at the carboxy terminus). These additionalhistidines (His-tag) decreases significantly (circa 50%) the catalytic activityof the protein. On the other hand, in the presence of lipid interfaces animportant superactivation (up to 100%) is also observed. Since at the workingpH 6.8 an important population of histidine residues are dissociated andconferred additional positive charges to the enzyme, we evaluated the abilityof negatively charged interfaces to modulate β-GalHis activity. Interestingly the superactivation exerted by DOPC:DOPG(80:20, molar ratio) MLVs is even greater than that induced by zwitterionicbilayers of pure DOPC. The molecular organization of those lipid mixtures atthe air-water interface served as membrane models and were studied throughsurface pressure?mean molecular area compression isotherms. Monolayers werecomposed of pure DOPC, pure DOPG or DOPC:DOPG mixtures at different molarratios (80:20, 60:40, 40:60 and 20:80). Mixtures showed a non-ideal behaviorwith positive deviation from ideality (repulsive interaction between thecomponents). Our results suggest that, beyond the attractive electrostacticinteractions between the positively charged His tag and the negatively chargedsurface, the higher sensitivity to the negative interface is being favored bythe reduction of the surface free energy associated to the β-GalHis?membrane binding. This contributesto an increased surface density of β-GalHis at the membrane which translates into an increased specificactivity.