Poster Session II - Abstract # 6
Characterizing the Folding Pathway of TolC, an Outer membrane Component of Antibiotic Efflux Pumps
Ayotunde P. Ikujuni1, S. Jimmy Budiardjo2, Emre Firlar3 , Andrés Cordova1, Jason T. Kaelber3 and Joanna S.G. Slusky1,2
1Department of Molecular Biosciences and 2Center for Computational Biology, The University of Kansas, Lawrence, KS, USA; 3Rutgers New Jersey CryoEM/CryoET Core Facility and Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
Overexpression of tripartite efflux pump systems in gram-negative bacteria has been reported to be linearly correlated with antibiotic resistance in clinical isolates as well as in selected mutants of different bacterial pathogens. The outer membrane subunit of the efflux pump, TolC, is a structurally unique outer membrane protein and hence difficult to produce at a sufficient yield for biochemical characterization. We have developed a method of refolding by which TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. Using circular dichroism, thermal denaturation, conformation assay and disulfide crosslinking, we found that the folding stages of TolC include the formation of periplasmic intermediates. We anticipate that our findings will help in developing better efflux pumps inhibitors.