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Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...
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The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
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Detergent headgroups control TolC folding in vitro.

Ayotunde Paul Ikujuni1, S Jimmy Budiardjo2, Rik Dhar1

  • 1Department of Molecular Biosciences, The University of Kansas, Lawrence, Kansas.

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|February 11, 2023
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Detergent micelles influence the in vitro folding of TolC, an essential antibiotic efflux pump component. Nonionic detergents promote functional trimeric TolC formation, crucial for understanding and combating antibiotic resistance.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • TolC is the outer membrane protein of the Escherichia coli efflux pump system, mediating antibiotic expulsion.
  • Overexpression of efflux pumps in bacterial pathogens reduces antibiotic susceptibility.
  • Characterizing membrane proteins like TolC is vital for understanding efflux mechanisms and developing new therapeutics.

Purpose of the Study:

  • To investigate the in vitro folding of TolC into its functional trimeric state from inclusion bodies.
  • To determine the effect of detergent micelle headgroup composition on TolC folding.
  • To identify optimal conditions for producing active TolC for further studies.

Main Methods:

  • Recombinant TolC protein was produced and purified from inclusion bodies.
  • In vitro refolding experiments were conducted using various detergents with different headgroup compositions and alkyl chain lengths.
  • The oligomeric state and functional folding of refolded TolC were assessed using biophysical techniques.

Main Results:

  • The in vitro folding of TolC into its functional trimeric form is critically dependent on the detergent micelle headgroup composition.
  • Nonionic detergents promote the formation of functional trimeric TolC.
  • Zwitterionic detergents lead to the formation of non-native, oligomeric TolC structures, while shorter alkyl chain nonionic detergents enhance folding.

Conclusions:

  • Detergent choice is a key factor in achieving successful in vitro refolding of the trimeric TolC protein.
  • Understanding these detergent-mediated folding pathways is essential for structural and biophysical characterization of TolC.
  • Further research is needed to explore the impact of lipid headgroup charges on TolC folding in biological membranes.