From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Summary

β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Abstract

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite β-barrel outer membrane protein (OMP), general protocols are presented for the production of target β-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

Introduction

β-barrel OMPs can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria^1-3. While they serve similar roles as α-helical proteins, they have a very different fold consisting of a central membrane-embedded β-barrel domain ranging from 8-26 anti-parallel β-strands with each strand being intimately connected to the two neighboring strands (Figures 1 and 2). The first and last strands of the β-barrel domain then interact with one another, almost exclusively in an anti-parallel fashion (except for mitochondrial VDAC), to close and seal the β-barrel domain from....

Protocol

1. Cloning and Expression

Note: To enable structural studies, sufficient quantities of highly purified protein must be prepared, and this generally starts with the cloning and overexpression of the target β-barrel outer membrane protein (OMP) in E. coli (Figure 4). To date, all β-barrel OMP structures, including those structures for mitochondrial VDAC, have been derived from bacterially expressed protein¹¹. Here, general protocols are presented for cloning and expressing β-barrel OMPs for (1) native expression directly into bacterial membranes and (2) expression into inclusions bodies for

Results

YiuR is a TonB dependent iron transporter that is a putative vaccine target against Yersinia pestis. It was originally identified using a microarray assay. Here, the steps that were taken to determine the structure of YiuR using X-ray crystallography are outlined (Figure 9). For cloning, the DNA sequence of YiuR (minus the N-terminal signal sequence) was PCR amplified from genomic DNA and subcloned into a vector containing an N-terminal pelB signal sequence and 1.......

Discussion

β-barrel OMPs serve essential roles in Gram-negative bacteria, mitochondria and chloroplasts and are important targets for structural analysis that offer a wealth of information about essential molecular mechanisms at the outer membranes of these respective organelles. However, producing enough sample for structural analysis is not always straightforward and therefore, a general pipeline is presented for the production of sufficient quantities of target β-barrel OMPs for structure determination, explaining in d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Crystallization Robot	TTP Labtech, Art Robbins	-	Any should work here, except for LCP crystallization
PCR thermocycler	Eppendorf, BioRad	-
Media Shaker	New Brunswick, Infors HT	-
UV-vis spectrometer	Eppendorf	-
SDS-PAGE apparatus	BioRad	1645050, 1658005
SDS-PAGE and native gels	BioRad, Life Technologies	4561084, EC6035BOX (BN1002BOX)
AkTA Prime	GE Healthcare	-
AkTA Purifier	GE Healthcare	-
Microcentrifuge	Eppendorf	-
Centrifuge (low-medium speed)	Beckman-Coulter	-
Ultracentrifuge (high speed)	Beckman-Coulter	-
SS34 rotor	Sorvall	-
Type 45 Ti rotor	Beckman-Coulter	-
Type 70 Ti rotor	Beckman-Coulter	-
Dounce homogenizer	Fisher Scientific	06 435C
Emulsiflex	Avestin	-
Dialysis tubing	Sigma	D9652
LCP tools	Hamilton, TTP Labtech	-
VDX 24 well plates	Hampton Research	HR3-172
Sandwich plates	Hampton Research, Molecular Dimensions	HR3-151, MD11-50 (MD11-53)
Grace Crystallization sheets	Grace Bio-Labs	875238
HiPrep S300 HR column	GE Healthcare	17-1167-01
Q-Sepharose column	GE Healthcare	17-0510-01
Crystallization screens	Hampton Research, Qiagen, Molecular Dimensions	-
Gas-tight syringe (100 ml)	Hamilton