Biophysical description of the Thermoalkali-philic Molecular Motor reveals a high torque implying an energetically conservative enzyme
Seminarroom PH 3024
F1FoATP synthases are bidirectional molecular motors that translocate protons across the membrane barrier by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acidin/alkalineout), challenging Mitchell’s chemiosmotic model. Unlike most mesophilic ATP synthases, alkaliphilic enzymes have a tightly regulated ATP hydrolysis activity, which can be relieved in the presence of LDAO . To provide a platform for understanding the mechanical aspects of this regulation, we characterized the rotary dynamics of TA2F1, a thermoalkaliphilic ATPsynthase from Caldalkalibacillus thermarum TA2.A1 with two robust forms of single-molecule analysis, a magnetic bead or a gold nanoparticle. TA2F1 rotated in a counter-clockwise direction in both systems, adhering to Michealis-Menten kinetics with a maximum rotation rate (Vmax) of 112.4 revolutions/s. Observation of rotation in the absence of LDAO or without mechanical stimulation by magnetic tweezers was both rare and brief. Using a gold nanoparticle TAF1 showed three rotational pauses at all ATP concentrations tested, each separated by angle of 120°, however, close to Km the 120º steps were further resolved into six sub-steps. TAF1 co-purifies with 2 ADP, leading to the suggestion of an adjusted reaction scheme for this enzyme where inorganic phosphate binds in the site of previously bound ADP. Torque measurements revealed the highest torque, 52pN, observed for an F1 molecule using fluctuation theorem. The mechanism of LDAO activation and implications of a high torque in terms of extreme environment adaptation are presented.
1. D.G.G. McMillan, S. Keis, P. Dimroth, G.M. Cook, A specific adaptation in the a-subunit of thermoalkaliphilic F1FO-ATP synthase enables ATP synthesis at high pH but not at neutral pH values 282 (2007) 17395-17404