Name: from constructs to crystals &#8211; towards structure determination of &#946;-barrel outer membrane proteins
Uploaded: 2016-07-04T13:00:00
Description: Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

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.).

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 &#946;-barrel outer membrane protein (OMP) in E. coli (Figure 4). To date, all &#946;-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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&#946;-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 &#946;-barrel OMPs for structure determination, explaining in d...

&#946;-barrel OMPs can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria1-3. While they serve similar roles as &#945;-helical proteins, they have a very different fold consisting of a central membrane-embedded &#946;-barrel domain ranging from 8-26 anti-parallel &#946;-strands with each strand being intimately connected to the two neighboring strands (Figures 1 and 2). The first and last strands of the &#946;-barrel domain then interact with one another, almost exclusively in an anti-parallel fashion (except for mitochondrial VDAC), to close and seal the &#946;-barrel domain from the surrounding membrane. All &#946;-barrel OMPs have extracellular loops of varying sequence and length which play an important role in ligand interactions and/or protein-protein contacts, with these loops sometimes being as large as 75 residues, such as found in Neisserial transferrin binding protein A (TbpA)4. &#946;-barrel OMPs can also have N-terminal or C-terminal periplasmic extensions which serve as additional domains for the protein&#39;s functional purpose (e.g., BamA5-7, FimD8,9, FadL10). While many types of &#946;-barrel OMPs exist11, two of the more common types are described below as examples for those less familiar with the field, (1) TonB-dependent transporters and (2) autotransporters.
TonB-dependent transporters (e.g., FepA, TbpA, BtuB, Cir, etc.) are essential for nutrient import and contain an N-terminal plug domain consisting of ~150 residues that is found tucked inside a C-terminal 22-stranded &#946;-barrel domain embedded into the outer membrane12 (Figure 3). While this plug domain prevents substrate from freely passing through the barrel domain, substrate binding induces a conformational change within the plug domain that leads to pore formation (either by plug rearrangement or by partial/full ejection of the plug) which can then facilitate substrate transport across the outer membrane into the periplasm. TonB-dependent transporters are especially important for the survival of some pathogenic strains of Gram-negative bacteria such as Neisseria meningitidis that have evolved specialized transporters that hijack nutrients such as iron directly from human host proteins4,13,14.
Autotransporters belong to the type V secretion system of Gram-negative bacteria and are &#946;-barrel OMPs that consist of a &#946;-barrel domain (typically 12-strands as with EstA and EspP) and a passenger domain that is either secreted or presented at the surface of the cell15,16 (Figure 3). These &#946;-barrel OMPs often serve important roles in cell survival and virulence with the passenger domain serving either as a protease, adhesin, and/or other effector that mediates pathogenesis.
Structural methods such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM) allow us to determine models for the OMPs at atomic resolution which can in turn be used to decipher exactly how they function within the outer membrane. This invaluable information may then be used for drug and vaccine development if applicable. For example, transferrin binding protein A (TbpA) is found on the surface of Neisseria and is required for pathogenesis because it directly binds human transferrin and then extracts and imports the iron for its own survival. Without TbpA, Neisseria cannot scavenge iron from the human host and are rendered non-pathogenic. After the crystal structure of human transferrin bound to TbpA4 was solved, it became much clearer how the two proteins associated, what regions of TbpA mediated the interaction, what residues were important for iron extraction by TbpA, and how one might develop therapeutics against Neisseria targeting TbpA. Therefore, given the importance of &#946;-barrel OMPs in Gram-negative bacteria for survival and pathogenesis, as well as in mitochondria and chloroplast function, and the need for additional structural information about this unique class of membrane proteins and the systems in which they function, general protocols are presented with the overall goal of expressing and purifying target OMPs at high levels for characterization by structural methods.

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			<td height="40" style="height:40px;width:175px;">Crystallization Robot</td>
			<td style="width:159px;">TTP Labtech, Art Robbins</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;">Any should work here, except for LCP crystallization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PCR thermocycler</td>
			<td style="width:159px;">Eppendorf, BioRad</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Media Shaker</td>
			<td style="width:159px;">New Brunswick, Infors HT</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">UV-vis spectrometer</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SDS-PAGE apparatus</td>
			<td style="width:159px;">BioRad</td>
			<td style="width:227px;">1645050, 1658005</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">SDS-PAGE and native gels</td>
			<td style="width:159px;">BioRad, Life Technologies</td>
			<td style="width:227px;">4561084, EC6035BOX (BN1002BOX)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Prime</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Purifier</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microcentrifuge</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Centrifuge (low-medium speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Ultracentrifuge (high speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SS34 rotor</td>
			<td style="width:159px;">Sorvall</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 45 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 70 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dounce homogenizer</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:227px;">06 435C</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Emulsiflex</td>
			<td style="width:159px;">Avestin</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dialysis tubing</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:227px;">D9652</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">LCP tools</td>
			<td style="width:159px;">Hamilton, TTP Labtech</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">VDX 24 well plates</td>
			<td style="width:159px;">Hampton Research</td>
			<td style="width:227px;">HR3-172</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Sandwich plates</td>
			<td style="width:159px;">Hampton Research, Molecular Dimensions</td>
			<td style="width:227px;">HR3-151, MD11-50 (MD11-53)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Grace Crystallization sheets</td>
			<td style="width:159px;">Grace Bio-Labs</td>
			<td style="width:227px;">875238</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HiPrep S300 HR column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-1167-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Q-Sepharose column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-0510-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:175px;">Crystallization screens</td>
			<td style="width:159px;">Hampton Research, Qiagen, Molecular Dimensions</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Gas-tight syringe (100 mL)</td>
			<td style="width:159px;">Hamilton&#160;</td>
			<td style="width:227px;">????</td>
			<td></td>
		</tr>
	</tbody>
</table>

from constructs to crystals &#8211; towards structure determination of &#946;-barrel outer membrane proteins

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, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-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 &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-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.

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...

Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

&#946;-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 &#946;-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% ...

The overall goal of this method is to obtain milligram quantities of outer membrane beta-barrel proteins that will crystallize and defract. This method can help answer key questions in the membrane protein folding and transport field, such as how beta-barrel proteins are inserted in cell membranes. The main advantage of this technique is that it is robust for beta-barrel proteins.Generally, individuals new to this method will struggle, because choice of detergents for purification, crystallization, and defraction is challenging. Jeremy Guerin, a postdoc from Susan Buchanan's laboratory, will be demonstrating the procedure, in addition to myself and colleagues in the lab. Begin this procedure with construction of a T7 expression vector containing the codon optimized target outer membrane protein, or OMP gene, for in vivo expression to the membrane as described in the text protocol.Transform the construct into an expression strain of bacteria for expression by pipetting one microliter of plasmid into 50 microliters of BL21(DE3)chemically competent cells, and mix gently by pipetting up and down. Incubate on ice for 30 minutes. Following incubation, heat pulse at 42 degrees Celsius for 30 seconds using a water bath, and then place back on ice for one minute.Add one milliliter of pre-warmed SOC media and shake at 37 degrees Celsius for one hour at 1000 RPM using a bench-top shaker incubator. Following incubation, plate 100 microliters of cells onto LB Agar plates containing an appropriate antibiotic and incubate overnight at 37 degrees Celsius, inverted. Next, perform small-scale expression tests by inoculating five milliliters of LB containing antibiotic culture with a single colony.Repeat for five to 10 colonies. Grow at 37 degrees Celsius with shaking to an optical density at 600 nanometers of approximately zero point six. Induce expression of the target OMP by adding five microliters of one molar IPTG to each culture tube and allow to grow an additional one to two hours.To compare expression levels for all the colonies, centrifuge one milliliter of each culture for one minute at 15, 000 times G, using a microcentrifuge. Remove the supernatant and resuspend the cells in 200 microliters of 1X SDS-PAGE loading buffer. Heat at 100 degrees Celsius for five minutes, and then centrifuge again at 15, 000 times G for five minutes.Analyze the samples using SDS-PAGE by pipetting 20 microliters into each well of a ten per cent gel. Run the gel for 35 minutes at a constant 200 volts. Finally, harvest the cells by centrifugation at 6000 times G for ten minutes.Resuspend the cells in lysis buffer at a ratio of five milliliters per gram of cell paste. Lyse the cells using a French press or cell homogenizer. Then, spin the lysed cells at 15, 000 times G for 30 minutes at four degrees Celsius to remove unlysed cells and cell debris.Transfer the supernatant to a clean tube and centrifuge again at high speed for one hour at four degrees Celsius. The resulting pellet is the membrane fraction which contains the protein of interest. Using a Dounce homogenizer, resuspend the membrane fraction.First, transfer the membranes and then add solubilization buffer at 2X concentration without detergent. Pour the resuspended membranes into a graduated cylinder and add water until a final concentration of 1X solubilization buffer is reached. Transfer the sample to a beaker and add detergent slowly to a final concentration of approximately 10 times the critical micelle concentration.Then, stir for 0.5 to 16 hours at four degrees Celsius, depending on how easily the target protein is extracted from the membranes. Finally, centrifuge the solubilized sample at 300, 000 times G for one hour at four degrees Celsius. Prepare a one to five milliliter IMAC column or use a pre-packed column.Perform subsequent chromatography steps at four degrees Celsius. Flush with water to remove any traces of preservatives. If using an automated purification system, install the column according to manufacturer's instructions.Prepare 500 milliliters of IMAC buffer A without imidizole and 250 millilliters of IMAC buffer B with one molar imidizole. Equilibrate the IMAC column using 10 column volumes of IMAC buffer A.Add imidizole to the protein sample to a final concentration of 25 millimolar and mix. Then, load the sample onto the equilibrated IMAC column at two milliliters per minute.Collect the flow-through. Next, wash the IMAC column with five column volumes each of increasing concentrations of imidizole, using buffer B.Collect the washes in two milliliter fractions. Elute the sample with a final concentration of 250 millimolar for five column volumes and collect the eluted sample in two milliliter fractions.Analyze the flow-through, the wash fractions, and the elution fractions using SDS-PAGE analysis based on their absorbance at 280 nanometers. Pool the fractions containing the target beta-barrel OMP as verified by SDS-PAGE analysis. Then, remove the 6X histadine tag by adding TEV protease to the pooled sample and incubating overnight at four degrees Celsius with gentle rocking.Load the sample protease solution onto an IMAC column again to separate the target beta-barrel OMP from the cleaved tags and any uncleaved sample. The flow-through will contain the cleaved sample lacking the tag. To prepare for crystallization, load the sample onto a gel filtration column into a buffer containing the detergent to be used for crystallization.Collect one milliliter fractions and analyze using SDS-PAGE analysis based on their absorbance at 280 nanometers. Pool those fractions containing the target protein, and then concentrate to approximately 10 milligrams per milliliter. Prior to preparing samples, assemble the gel apparatus.Use pre-cooled gel running buffer or run the gel in the cold room. Filter the sample using a 0.22 micron centrifugal filter to remove particulates and precipitation. Then, pipette 0.25 microliters of the sample into two 1.5 milliliter micro centrifuge tubes.Label one as boiled"and the other as RT"Next, add 9.75 microliters of the sample buffer to each tube and mix by pipetting. To both samples, also add 10 microliters of 2X SDS loading buffer and mix gently by pipetting. Boil the boiled"sample at 95 degrees Celsius for five minutes, while leaving the RT"sample at room temperature.Then, spin the boiled"sample briefly. Next, load 20 microliters of both samples onto the pre-assembled native gel. After running the gel for 60 minutes at a constant 150 volts remove the gel and soak in staining solution to visualize the results.Proceed with crystallization trials in detergent, bi-cells, and lipidic cubic phase, using previously published Jove methods. Crystallization heads are visualized under a UV microscope to verify that the crystals are, indeed, protein and not solved, lipid or detergent. Lead crystals are screened using a home source or at the synchrotron to see if the crystals defract.After data collection of crystals that defract well, process the data using processing software, such as HKL2000. Transfer resulting files to a software suite for structure determination, such as Phoenix Suit, and perform molecular replacement for initial phasing and structure determination. The crystal structure of YIUR was determined at 2.6 Angstrom resolution using this method.Once mastered, this technique can be done in two weeks, if it is performed properly. After watching this video, you should have a good understanding of how to express and purify bacterial outer membrane proteins for structural studies.

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