Ikujuni, A. P. | Search bioRxiv

bioRxiv2022

Detergent headgroups control TolC folding in vitro

TolC is the trimeric outer membrane component of the efflux pump system in E. coli responsible for antibiotic efflux from bacterial cells. Over-expression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which bioactive proteins are recovered. Here we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native trimeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

biophysics↗

bioRxiv2020

High yield preparation of outer-membrane protein efflux pumps by in vitro refolding is concentration dependent

Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

biophysics↗