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Chemistry

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

Published: July 4th, 2016

DOI:

10.3791/53245

1Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 3National Institute of General Medical Sciences (NIGMS), National Institutes of Health

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.

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.

β-barrel OMPs can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria1-3. 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....

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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 .......

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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.......

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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.......

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We would like to thank Herve Celia of the CNRS for providing the UV images and Chris Dettmar and Garth Simpson in the Department of Chemistry at Purdue University for providing the SONICC images. We would like to acknowledge funding from the National Institute of Diabetes and Digestive and Kidney Diseases and the Intramural Research Program at the National Institutes of Health. Additionally, we would like to acknowledge additional funding from the National Institute of General Medical Sciences (A.M.S. and C.J.), National Institute of Allergy and Infectious Diseases (N.N. 1K22AI113078-01), and the Department of Biological Sciences at Purdue University (N.N.).

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NameCompanyCatalog NumberComments
Crystallization RobotTTP Labtech, Art Robbins-Any should work here, except for LCP crystallization
PCR thermocyclerEppendorf, BioRad-
Media ShakerNew Brunswick, Infors HT-
UV-vis spectrometerEppendorf-
SDS-PAGE apparatusBioRad1645050, 1658005
SDS-PAGE and native gelsBioRad, Life Technologies4561084, EC6035BOX (BN1002BOX)
AkTA PrimeGE Healthcare-
AkTA PurifierGE Healthcare-
MicrocentrifugeEppendorf-
Centrifuge (low-medium speed)Beckman-Coulter-
Ultracentrifuge (high speed)Beckman-Coulter-
SS34 rotorSorvall-
Type 45 Ti rotorBeckman-Coulter-
Type 70 Ti rotorBeckman-Coulter-
Dounce homogenizerFisher Scientific06 435C
EmulsiflexAvestin-
Dialysis tubingSigmaD9652
LCP toolsHamilton, TTP Labtech-
VDX 24 well platesHampton ResearchHR3-172
Sandwich platesHampton Research, Molecular DimensionsHR3-151, MD11-50 (MD11-53)
Grace Crystallization sheetsGrace Bio-Labs875238
HiPrep S300 HR columnGE Healthcare17-1167-01
Q-Sepharose columnGE Healthcare17-0510-01
Crystallization screensHampton Research, Qiagen, Molecular Dimensions-
Gas-tight syringe (100 mL)Hamilton ????

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