Structure of Voltage-Dependent Anion Channel-Tethered Bilayer Lipid Membranes Determined using Neutron Reflectivity

Published

December 1, 2018

Author(s)

David Paul Hoogerheide, Sergei Yu Noskov, Adam J. Kuszak, Tatiana K. Rostovtseva, Hirsh Nanda

Abstract

Neutron reflectivity (NR) has emerged as a powerful technique to study the structure and behavior of membrane proteins at planar lipid interfaces. Integral membrane proteins (IMPs) remain a significant challenge for NR due to the difficult of forming complete bilayers with sufficient protein density for scattering techniques. One strategy to achieve high protein density on a solid substrate is the capture of detergent-stabilized, affinity-tagged IMPs on a nitrilotriacetic acid (NTA)-functionalized self-assembled monolayer (SAM), followed by reconstitution into the lipids of interest. Such protein-tethered bilayer lipid membranes (ptBLMs) have the notable advantage of uniform IMP orientation on the substrate. We use NR to provide a structural characterization of the ptBLM process from the formation of the SAM, the capture of the detergent-stabilized IMP, and the lipid reconstitution. WE use the mitochondrial outer membrane voltage-dependent anion channel (VDAC), which controls the exchange of bioenergetic metabolites between mitochondria and the cytosol, as a model β-barrel IMP. Molecular dynamics simulations are used for comparison to the experimental results and to inform the parameters of the physical models describing the NR data. We show that the detailed structure of the SAM depends on the density of the NTA cheating groups; measure the relative content of detergent and protein in surface-immobilized detergent-stabilized VDAC; show that the lipid bilayer is complete to within a few percent, using the known atomic structure of VDAC; and deomonstrate the presence of excess lipid above the reconstituted bilayer that is of consequence for more indirect structural and functional studies.

Citation

Acta Crystallographica Section D-Biological Crystallography

Volume

D74

Issue

12

Pub Type

Journals

Download Paper

Local Download

Keywords

neutron reflectivity, voltage-dependent anion channel, protein-tethered bilayer lipid membranes, affinity purification, molecular dynamics simulations, voltage gating of ion channels

Citation

, D. , , S. , , A. , , T. and Nanda, H. (2018), Structure of Voltage-Dependent Anion Channel-Tethered Bilayer Lipid Membranes Determined using Neutron Reflectivity, Acta Crystallographica Section D-Biological Crystallography, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=925397 (Accessed December 17, 2024)