Abstract
Type I secretion systems (T1SS) are ubiquitous transport machineries in Gram-negative bacteria. They comprise a relatively simple assembly of three membrane-localised proteins: an inner-membrane complex composed of an ABC transporter and a membrane fusion protein, and a TolC-like outer membrane component. T1SS transport a wide variety of substrates with broad functional diversity. The ABC transporter hemolysin B (HlyB), for example, is part of the hemolysin A-T1SS in Escherichia coli. In contrast to canonical ABC transporters, an accessory domain, a C39 peptidase-like domain (CLD), is located at the N-terminus of HlyB and is essential for secretion. In this study, we have established an optimised purification protocol for HlyB and the subsequent reconstitution employing the saposin-nanoparticle system. We point out the negative influence of free detergent on the basal ATPase activity of HlyB, studied the influence of a lysolipid or lipid matrix on activity and present functional studies with the full-length substrate proHlyA in its folded and unfolded states, which both have a stimulatory effect on the ATPase activity.
Highlights
ObjectivesIn contrast to other studies conducted with detergent-purified HlyB where a shortened version of the natural substrate was used in the ATPase assays, we aimed to employ the full-length, yet hemolytically inactive precursor form of HlyA, proHlyA
We demonstrated that the properties of a lipid bilayer can influence stimulation of an ABC transporter by its substrate, and that this is not necessarily reflected by a change in the basal ATPase activity
We established a protocol to embed the ABC transporter HlyB in detergent-derived lipoprotein particles and point out that the presence of free detergent micelles can affect the functionality of a membrane protein
Summary
In contrast to other studies conducted with detergent-purified HlyB where a shortened version of the natural substrate was used in the ATPase assays, we aimed to employ the full-length, yet hemolytically inactive precursor form of HlyA, proHlyA
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have