Magnetically Directed Two-Dimensional Crystallization of OmpF Membrane Proteins in Block Copolymers

Abstract

Two-dimensional (2D) alignment and crystallization of membrane proteins (MPs) is increasingly important in characterizing their three-dimensional (3D) structure, in designing pharmacological agents, and in leveraging MPs for biomimetic devices. Large, highly ordered MP 2D crystals in block copolymer (BCP) matrices are challenging to fabricate, but a facile and scalable technique for aligning and crystallizing MPs in thin-film geometries would rapidly translate into applications. This work introduces a novel method to grow larger and potentially better ordered 2D crystals by performing the crystallization process in the presence of a strong magnetic field. We demonstrate the efficacy of this approach using a β-barrel MP, outer membrane protein F (OmpF), in short-chain polybutadiene-poly(ethylene oxide) (PB-PEO) membranes. Crystals grown in a magnetic field were up to 5 times larger than conventionally grown crystals, and a signal-to-noise (SNR) analysis of diffraction peaks in Fourier transforms of specimens imaged by negative-stain electron microscopy (EM) and cryo-EM showed twice as many high-SNR diffraction peaks, indicating that the magnetic field also improves crystal order.

Computing the Diamagnetic Susceptibility and Diamagnetic Anisotropy of Membrane Proteins from Structural Subunits

“…For each case we highlight the calculated value for a single beta barrel protein, OmpF, and a single alpha helical protein, Aqp0, to draw parallels with our previous experimental work. 13 The calculated DA values of the 628 MPs (including the monotopic structures) investigated in this study are individually reported in SI Table S3.…”

“…[5][6][7][8][9][10][11][12] A promising route toward manipulating the crystallization process is to apply an external magnetic field during MP self-assembly. 13 This technique was used in crystallization of Aquaporin-0 (Aqp0), a water pore expressed in the fiber cells of the mammalian lens, and Outer membrane protein F (OmpF), a porin expressed in Escherichia coli that allows for the passive diffusion of hydrophilic small molecules. We observed significant improvements in both short-and long-range order for both proteins, though the effect was stronger for the OmpF than for Aqp0.…”

Stoichiometry determination of macromolecular membrane protein complexes

“…Both electron and X-ray crystallography depend on the successful crystallization of proteins and protein complexes. There have been many improvements in the production of membrane proteins for crystallization (Clark et al, 2011;Schlegel et al, 2014) and the formation and stabilization of crystals (Carpenter et al, 2008;Klara et al, 2016). However, as seen in crystal structures of components of the TtATPase, different subunits of type III secretion systems, as well as the nuclear pore complex (Lee et al, 2010;Worrall et al, 2010;Stuwe et al, 2015), mostly structures of soluble components or extramembrane domains of transmembrane proteins have been solved for large membrane-spanning complexes.…”

Porous Vesicles with Extrusion‐Tunable Permeability and Pore Size from Mixed Solutions of PEO–PPO–PEO Triblock Copolymers

“…Depending on the ratio of the hydrophilic and hydrophobic block volume fractions, packing constraints lead to structures such as (in order of decreasing hydrophilic fraction) spherical micelles, wormlike micelles, membranes/vesicles, and inverted microstructures . Because these polymers have greater chemical and mechanical stability than lipids, and because some BCs have been used to incorporate transmembrane proteins, as well as drugs, DNA, dyes, and nanoparticles, BC membranes are widely studied as lipid‐membrane mimics with the stability required for applications such as sensors, separations, energy production, imaging, and drug and gene delivery …”