sample-preparation-using-lipid-monolayer-method-for-electron

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

Arizona State University

Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. All available structures from the genus Borrelia represent proteins expressed without a membrane-targeting signal peptide, thus avoiding conserved pathways that modify, fold and assemble membrane protein complexes. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. P66 was also expressed using the PelB signal sequence fused to maltose binding protein. Membrane-associated BBA57 lipoprotein was solubilized by non-ionic and zwitterionic detergents. We show BBA57 translocation to the outer membrane, purification at a level sufficient for structural studies, and evidence for an α-helical multimer. Previous studies showed multiple critical roles of BBA57 in transmission, joint arthritis, carditis, weakening immune responses, and regulating other Bb outer surface proteins. In describing the first purification of membrane-translocated BBA57, this work will support subsequent studies that reveal the precise mechanisms of this important Lyme disease virulence factor.

Membrane directed expression in Escherichia coli of BBA57 and other virulence factors from the Lyme disease agent Borrelia burgdorferi.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Membrane directed expression in Escherichia coli of BBA57 and other virulence factors from the Lyme disease agent Borrelia burgdorferi.

Chloe D. Truong

School of Molecular Sciences,
Center for Applied Structural Discovery,
Biodesign Institute,
Center for Applied Structural Discovery, Biodesign Institute

Arizona State University

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Chloe D. Truong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Molecular Sciences,Center for Applied Structural Discovery,Biodesign Institute,Center for Applied Structural Discovery, Biodesign Institute

Arizona State University

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

School of Molecular Sciences,
Center for Applied Structural Discovery,
Biodesign Institute,
Center for Applied Structural Discovery, Biodesign Institute