Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane

Yue Ding, Takuya Shiota, Anton P. Le Brun, Rhys A. Dunstan, Bo Wang, Hsien-Yi Hsu, Trevor Lithgow, Hsin-Hui Shen

Research output: Contribution to journalArticleResearchpeer-review

Abstract

In Gram–negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-L-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.

Original languageEnglish
Article number183317
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1862
Issue number9
DOIs
Publication statusPublished - 1 Sep 2020

Keywords

  • Membrane biogenesis
  • Neutron reflectometry
  • Outer membrane protein
  • β-barrel assembly machinery

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