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.).

<ol>
	<li>Walther, D. M., Rapaport, D., Tommassen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+beta-barrel+membrane+proteins+in+bacteria+and+eukaryotes:+evolutionary+conservation+and+divergence.">Biogenesis of beta-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence.</a> Cell Mol Life Sci. 66, 2789-2804 (2009).</li><li>Ricci, D. P., Silhavy, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bam+machine:+A+molecular+cooper.">The Bam machine: A molecular cooper.</a> Biochim Biophys Acta. 1818, 1067-1084 (2012).</li><li>Hagan, C. L., Silhavy, T. J., Kahne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Barrel+membrane+protein+assembly+by+the+Bam+complex.">beta-Barrel membrane protein assembly by the Bam complex.</a> Ann Rev of Biochem. 80, 189-210 (2011).</li><li>Noinaj, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+iron+piracy+by+pathogenic+Neisseria.">Structural basis for iron piracy by pathogenic Neisseria.</a> Nature. 483, 53-58 (2012).</li><li>Noinaj, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insight+into+the+biogenesis+of+beta-barrel+membrane+proteins.">Structural insight into the biogenesis of beta-barrel membrane proteins.</a> Nature. 501, 385-390 (2013).</li><li>Gatzeva-Topalova, P. Z., Walton, T. A., Sousa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+YaeT:+conformational+flexibility+and+substrate+recognition.">Crystal structure of YaeT: conformational flexibility and substrate recognition.</a> Structure. 16, 1873-1881 (2008).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+an+essential+component+of+the+outer+membrane+protein+assembly+machine.">Structure and function of an essential component of the outer membrane protein assembly machine.</a> Science. 317, 961-964 (2007).</li><li>Geibel, S., Procko, E., Hultgren, S. J., Baker, D., Waksman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+energetic+basis+of+folded-protein+transport+by+the+FimD+usher.">Structural and energetic basis of folded-protein transport by the FimD usher.</a> Nature. 496, 243-246 (2013).</li><li>Phan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+FimD+usher+bound+to+its+cognate+FimC-FimH+substrate.">Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.</a> Nature. 474, 49-53 (2011).</li><li>van den Berg, B., Black, P. N., Clemons, W. M., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+long-chain+fatty+acid+transporter+FadL.">Crystal structure of the long-chain fatty acid transporter FadL.</a> Science. 304, 1506-1509 (2004).</li><li>Fairman, J. W., Noinaj, N., Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+biology+of+beta-barrel+membrane+proteins:+a+summary+of+recent+reports.">The structural biology of beta-barrel membrane proteins: a summary of recent reports.</a> Curr Op Struct Bio. 21, 523-531 (2011).</li><li>Noinaj, N., Guillier, M., Barnard, T. J., Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TonB-dependent+transporters:+regulation,+structure,+and+function.">TonB-dependent transporters: regulation, structure, and function.</a> Ann Rev of Micro. 64, 43-60 (2010).</li><li>Siburt, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hijacking+transferrin+bound+iron:+protein-receptor+interactions+involved+in+iron+transport+in+N.+gonorrhoeae.">Hijacking transferrin bound iron: protein-receptor interactions involved in iron transport in N. gonorrhoeae.</a> Metallomics. 1, 249-255 (2009).</li><li>Cornelissen, C. N., Hollander, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TonB-Dependent+Transporters+Expressed+by+Neisseria+gonorrhoeae.">TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae.</a> Front in Micro. 2 (117), (2011).</li><li>Dautin, N., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+in+gram-negative+bacteria+via+the+autotransporter+pathway.">Protein secretion in gram-negative bacteria via the autotransporter pathway.</a> Ann Rev of Micro. 61, 89-112 (2007).</li><li>Leo, J. C., Grin, I., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+V+secretion:+mechanism(s)+of+autotransport+through+the+bacterial+outer+membrane.">Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane.</a> Phil Trans of the Royal Soc of London. Series B, Biological Sciences. 367, 1088-1101 (2012).</li><li>Buchanan, S. K., Yamashita, S., Fleming, K. G., Engelman, E. H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comprehensive Biophysics. 5, 139-163 (2011).</li><li>Buchanan, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+colicin+I+receptor+bound+to+the+R-domain+of+colicin+Ia:+implications+for+protein+import.">Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.</a> EMBO J. 26, 2594-2604 (2007).</li><li>Yamashita, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+Ail-mediated+adhesion+in+Yersinia+pestis.">Structural insights into Ail-mediated adhesion in Yersinia pestis.</a> Structure. 19, 1672-1682 (2011).</li><li>Aslanidis, C., de Jong, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation-independent+cloning+of+PCR+products+(LIC-PCR).">Ligation-independent cloning of PCR products (LIC-PCR).</a> Nucleic Acids Res. 18, 6069-6074 (1990).</li><li>Haun, R. S., Serventi, I. M., Moss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+reliable+ligation-independent+cloning+of+PCR+products+using+modified+plasmid+vectors.">Rapid reliable ligation-independent cloning of PCR products using modified plasmid vectors.</a> BioTechniques. 13, 515-518 (1992).</li><li>Rosenbusch, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+major+envelope+protein+from+Escherichia+coli.+Regular+arrangement+on+the+peptidoglycan+and+unusual+dodecyl+sulfate+binding.">Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding.</a> J Biol Chem. 249, 8019-8029 (1974).</li><li>Barnard, T. J., Wally, J. L., Buchanan, S. K., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+integral+membrane+proteins.">Crystallization of integral membrane proteins.</a> Curr Prot in Prot Science. Chapter 17, Unit 17 19 (2007).</li><li>Ujwal, R., Abramson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+crystallization+of+membrane+proteins+using+the+lipidic+bicelle+method.">High-throughput crystallization of membrane proteins using the lipidic bicelle method.</a> JoVE. , e3383 (2012).</li><li>Faham, S., Ujwal, R., Abramson, J., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Aspects+of+Membrane+Proteins+Crystallization+in+Bicelles.">Practical Aspects of Membrane Proteins Crystallization in Bicelles.</a> Curr Topics in Membranes. 63, 111-127 (2009).</li><li>Li, D., Boland, C., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+robot+for+high-throughput+crystallization+of+membrane+proteins+in+lipidic+mesophases.">Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.</a> JoVE. , e4000 (2012).</li><li>Liu, W., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+lipidic+mesophases.">Crystallization of membrane proteins in lipidic mesophases.</a> JoVE. , (2011).</li><li>Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+refolding+of+an+80-kDa+iron+transporter+from+the+outer+membrane+of+Escherichia+coli.">Overexpression and refolding of an 80-kDa iron transporter from the outer membrane of Escherichia coli.</a> Biochem Soc Trans. 27, 903-908 (1999).</li><li>Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-barrel+proteins+from+bacterial+outer+membranes:+structure,+function+and+refolding.">Beta-barrel proteins from bacterial outer membranes: structure, function and refolding.</a> Curr Op in Struct Bio. 9, 455-461 (1999).</li><li>Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+process+of+folding+proteins+into+membranes:+challenges+and+progress.">The process of folding proteins into membranes: challenges and progress.</a> Arch of Biochem and Biophysics. 469, 46-66 (2008).</li><li>Pautsch, A., Schulz, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+outer+membrane+protein+A+transmembrane+domain.">Structure of the outer membrane protein A transmembrane domain.</a> Nature Struct Bio. 5, 1013-1017 (1998).</li><li>Cowan, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+OmpF+porin+in+a+tetragonal+crystal+form.">The structure of OmpF porin in a tetragonal crystal form.</a> Structure. 3, 1041-1050 (1995).</li><li>Ujwal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+mouse+VDAC1+at+2.3+A+resolution+reveals+mechanistic+insights+into+metabolite+gating.">The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating.</a> PNAS. 105, 17742-17747 (2008).</li><li>Robert, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+factor+Omp85+recognizes+its+outer+membrane+protein+substrates+by+a+species-specific+C-terminal+motif.">Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.</a> PLoS Bio. 4, e377 (2006).</li><li>Paramasivam, N., Habeck, M., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+C-terminal+insertional+signal+in+Gram-negative+bacterial+outer+membrane+proteins+species-specific+or+not?.">Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?.</a> BMC Genomics. 13, 510 (2012).</li><li>Agah, S., Faham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+membrane+proteins+in+bicelles.">Crystallization of membrane proteins in bicelles.</a> Methods in Mol Bio. 914, 3-16 (2012).</li><li>Ujwal, R., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+bicelles.">Crystallizing membrane proteins using lipidic bicelles.</a> Methods. 55, 337-341 (2011).</li><li>Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidic+cubic+phase+technologies+for+membrane+protein+structural+studies.">Lipidic cubic phase technologies for membrane protein structural studies.</a> Cur Op in Struct Bio. 21, 559-566 (2011).</li><li>Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P., Lanyi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacteriorhodopsin+at+1.55+A+resolution.">Structure of bacteriorhodopsin at 1.55 A resolution.</a> J Mol Biol. 291, 899-911 (1999).</li><li>Kato, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+channelrhodopsin+light-gated+cation+channel.">Crystal structure of the channelrhodopsin light-gated cation channel.</a> Nature. 482, 369-374 (2012).</li><li>White, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+agonist-bound+neurotensin+receptor.">Structure of the agonist-bound neurotensin receptor.</a> Nature. 490, 508-513 (2012).</li><li>Cherezov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Rasmussen, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+beta2+adrenergic+receptor-Gs+protein+complex.">Crystal structure of the beta2 adrenergic receptor-Gs protein complex.</a> Nature. 477, 549-555 (2011).</li><li>Haupert, L. M., Simpson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+protein+crystallization+trials+by+second+order+nonlinear+optical+imaging+of+chiral+crystals+(SONICC).">Screening of protein crystallization trials by second order nonlinear optical imaging of chiral crystals (SONICC).</a> Methods. 55, 379-386 (2011).</li></ol>

The authors declare that they have no competing financial interests.

&#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.

<table><tbody><tr><td>Crystallization Robot</td><td>TTP Labtech, Art Robbins</td><td>-</td><td>Any should work here, except for LCP crystallization</td></tr><tr><td>PCR thermocycler</td><td>Eppendorf, BioRad</td><td>-</td><td></td></tr><tr><td>Media Shaker</td><td>New Brunswick, Infors HT</td><td>-</td><td></td></tr><tr><td>UV-vis spectrometer</td><td>Eppendorf</td><td>-</td><td></td></tr><tr><td>SDS-PAGE apparatus</td><td>BioRad</td><td>1645050, 1658005</td><td></td></tr><tr><td>SDS-PAGE and native gels</td><td>BioRad, Life Technologies</td><td>4561084, EC6035BOX (BN1002BOX)</td><td></td></tr><tr><td>AkTA Prime</td><td>GE Healthcare</td><td>-</td><td></td></tr><tr><td>AkTA Purifier</td><td>GE Healthcare</td><td>-</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>-</td><td></td></tr><tr><td>Centrifuge (low-medium speed)</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Ultracentrifuge (high speed)</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>SS34 rotor</td><td>Sorvall</td><td>-</td><td></td></tr><tr><td>Type 45 Ti rotor</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Type 70 Ti rotor</td><td>Beckman-Coulter</td><td>-</td><td></td></tr><tr><td>Dounce homogenizer</td><td>Fisher Scientific</td><td>06 435C</td><td></td></tr><tr><td>Emulsiflex</td><td>Avestin</td><td>-</td><td></td></tr><tr><td>Dialysis tubing</td><td>Sigma</td><td>D9652</td><td></td></tr><tr><td>LCP tools</td><td>Hamilton, TTP Labtech</td><td>-</td><td></td></tr><tr><td>VDX 24 well plates</td><td>Hampton Research</td><td>HR3-172</td><td></td></tr><tr><td>Sandwich plates</td><td>Hampton Research, Molecular Dimensions</td><td>HR3-151, MD11-50 (MD11-53)</td><td></td></tr><tr><td>Grace Crystallization sheets</td><td>Grace Bio-Labs</td><td>875238</td><td></td></tr><tr><td>HiPrep S300 HR column</td><td>GE Healthcare</td><td>17-1167-01</td><td></td></tr><tr><td>Q-Sepharose column</td><td>GE Healthcare</td><td>17-0510-01</td><td></td></tr><tr><td>Crystallization screens</td><td>Hampton Research, Qiagen, Molecular Dimensions</td><td>-</td><td></td></tr><tr><td>Gas-tight syringe (100 ml)</td><td>Hamilton&amp;nbsp;</td><td></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 β-barrel Towards Structure Determination of Î²-barrel Outer Membrane Proteins | Chemistry | Video

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% ...

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13.4K Views.  Purdue University. 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.

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

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Immunology and Infection

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Bioengineering

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Multi-pass Transmembrane Proteins and &#946;-barrels

In multi-pass transmembrane proteins, the polypeptide chain crosses the membrane more than once. The transmembrane polypeptide chain either forms an &#945;-helix or &#946;-strand structure. &#945;-Helix containing multi-pass transmembrane proteins are ubiquitous, whereas &#946;-strand&#160;containing ones are mainly found in gram-negative bacteria, mitochondria, and chloroplasts.
&#945;-Helix containing multi-pass transmembrane proteins 
Multi-pass transmembrane proteins such as G-protein-linked receptors (GPCRs) and bacteriorhodopsin contain multiple transmembrane domains. All GPCRs have seven transmembrane &#945;-helices, but each receptor has its specific extracellular domain and G-protein-binding site. GPCRs bind a ligand and activate membrane protein called G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane.
Bacteriorhodopsin found in certain photosynthetic bacteria has seven transmembrane &#945;-helices. It is a light-driven proton pump that helps in ATP production by generating a proton gradient across the membrane.
&#946;-Strands containing multi-pass transmembrane proteins 
Some bacterial membrane proteins contain transmembrane &#946;-strands, which are arranged into a ring-like structure called the &#946;-barrel. A minimum of eight &#946;-strands are required to form the barrel-like structure where the edges of the two closely spaced &#946;-strands form hydrogen bonds together. The porin protein observed in the membrane of some gram-negative bacteria contains a transmembrane &#946;-barrel. Apart from bacteria, porin proteins are also found in mitochondrial and chloroplast membranes, which support the hypothesis that both mitochondria and chloroplast are evolved from prokaryotes.
Some part of this text is adapted from Openstax, Biology 2e, <a href="https://openstax.org/books/biology-2e/pages/9-1-signaling-molecules-and-cellular-receptors">Section 9.1: Signaling Molecules and Cellular Receptors</a>.

Multi-pass Transmembrane Proteins and &amp;#946;-barrels

In multi-pass transmembrane proteins, the polypeptide chain crosses the membrane more than once. The transmembrane polypeptide chain either forms an ...

Cell Biology

Unit 1

Membrane Structure and Components

KEY TERMS AND CONCEPTS

Structure of Porins

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and metabolites across the membrane. Mitochondrial porin precursors contain conserved amino acid sequences called beta signals at their C-terminal. Beta signals have a&#160; motif of PoXGXXHyXHy (Po-Polar, X-Any amino acid, G-Glycine, Hy-LargeHydrophobic), which are crucial for precursor recognition to initiate precursor assembly. Beta-barrel precursors move through the TOM complex on the outer mitochondrial membrane into the intermembrane space. The precursors interact with TIM chaperones inside the intermembrane space and are guided to a trimeric complex called the Sorting and Assembly machinery complex or SAM complex.&#160;
SAM complex has three components: a beta-barrel core called the SAM50 and accessory proteins SAM35 and SAM37. SAM50 comprises 16-beta-strands that span the outer membrane and expose an N-terminal polypeptide transport-associated domain or POTRA domain in the intermembrane space. The POTRA domain functions as a receptor for beta-barrel precursors and facilitates beta-strand assembly. In contrast, SAM50 interacts closely with accessory proteins SAM35/SAM37 towards the cytosolic side to help fold the beta-barrel precursors entering the intermembrane space.

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and ...

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

Summary

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Chapters in this video

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β₂-Microglobulin

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Multi-pass Transmembrane Proteins and β-barrels

Structure of Porins