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 Ã¢â‚¬â€œ Towards Structure Determination of ÃŽÂ²-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>
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		<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>
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		<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 Ã¢â‚¬â€œ Towards Structure Determination of ÃŽÂ²-barrel Outer Membrane Proteins at JoVE.com

From Constructs to Crystals ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ Towards Structure Determination of ÃƒÅ½Ã‚Â²-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.

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% ...

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