هناك العديد من الذين ساهموا في هذا العمل ومعظمها من الغشاء Caffrey الهيكلية والوظيفية المجموعة الأحياء، وكلاهما عضو في الماضي والحاضر. للجميع، وخاصة ليون وتان جينغ تشيوان يوسف، نتقدم بأحر الشكر والتقدير. وأيد هذا العمل في جزء من المنح المقدمة من مؤسسة العلوم ايرلندا (07/IN.1/B1836)، والمعاهد الوطنية للصحة (GM75915، وP50GM073210 U54GM094599)، وCOST FP7 وماري كوري الإجراءات (وPIEF CM0902-GA 2009- -235612).

<ol>
	<li>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=Crystallizing+membrane+proteins+for+structure+determination:+use+of+lipidic+mesophases.">Crystallizing membrane proteins for structure determination: use of lipidic mesophases.</a> Annu. Rev. Biophys. 38, 29-51 (2009).</li><li>Caffrey, M., Li, D., Dukkipati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+structure+determination+using+crystallography+and+lipidic+mesophases+-+recent+advances+and+successes.">Membrane protein structure determination using crystallography and lipidic mesophases - recent advances and successes.</a> Biochemistry. , (2012).</li><li>Caffrey, M., 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=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protocols. 4, 706-731 (2009).</li><li>Cherezov, V., Clogston, J., Papiz, M. Z., 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=Room+to+move:+crystallizing+membrane+proteins+in+swollen+lipidic+mesophases.">Room to move: crystallizing membrane proteins in swollen lipidic mesophases.</a> J. Mol. Biol. 357, 1605-1618 (2006).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., 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=A+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Setvens, R. C. <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>Chien, E. Y., Liu, W., Zhao, Q., Katritch, V., Han, G. W., Hanson, M. A., Shi, L., Newman, A. H., Javitch, J. A., Cherezov, V., Stevens, R. C. <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+human+dopamine+D3+receptor+in+complex+with+a+D2/D3+selective+antagonist.">Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist.</a> Science. 330, 1091-1095 (2010).</li><li>Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., Kobilka, B. 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+the+delta-opioid+receptor+bound+to+naltrindole.">Structure of the delta-opioid receptor bound to naltrindole.</a> Nature. 485, 400-404 (2012).</li><li>Haga, K., Kruse, A. C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W. I., Okada, T., Kobilka, B. K., Haga, T., Kobayashi, T. <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+human+M2+muscarinic+acetylcholine+receptor+bound+to+an+antagonist.">Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.</a> Nature. 482, 547-551 (2012).</li><li>Hanson, M. A., Roth, B., Jo, E., Griffith, M. T., Scott, F. L., Reinhart, G., Desale, H., Clemons, B., Cahalan, S. M., Schuerer, S. 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+a+lipid+G+protein-coupled+receptor.">Crystal structure of a lipid G protein-coupled receptor.</a> Science. 335, 851-855 (2012).</li><li>Jaakola, V. P., Griffith, M. T., Hanson, M. A., Cherezov, V., Chien, E. Y., Lane, J. R., Ijzerman, A. P., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2.6+angstrom+crystal+structure+of+a+human+A2A+adenosine+receptor+bound+to+an+antagonist.">The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.</a> Science. 322, 1211-1217 (2008).</li><li>Kruse, A. C., Hu, J., Pan, A. C., Arlow, D. H., Rosenbaum, D. M., Rosemond, E., Green, H. F., Liu, T., Chae, P. S., Dror, R. O. <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+dynamics+of+the+M3+muscarinic+acetylcholine+receptor.">Structure and dynamics of the M3 muscarinic acetylcholine receptor.</a> Nature. 482, 552-556 (2012).</li><li>Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., Pardo, L., Weis, W. I., Kobilka, B. K., Granier, S. <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+micro-opioid+receptor+bound+to+a+morphinan+antagonist.">Crystal structure of the micro-opioid receptor bound to a morphinan antagonist.</a> Nature. 485, 321-326 (2012).</li><li>Rasmussen, S. G., Choi, H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., Devree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I., Kobilka, B. 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+a+nanobody-stabilized+active+state+of+the+beta(2)+adrenoceptor.">Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor.</a> Nature. 469, 175-180 (2011).</li><li>Rasmussen, S. G. F., Devree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D. <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+&#946;2+adrenergic+receptor-Gs+protein+complex.">Crystal structure of the &#946;2 adrenergic receptor-Gs protein complex.</a> Nature. 477, 549-555 (2011).</li><li>Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Yao, X. J., Weis, W. I., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+engineering+yields+high-resolution+structural+insights+into+beta2-adrenergic+receptor+function.">GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.</a> Science. 318, 1266-1273 (2007).</li><li>Rosenbaum, D. M., Zhang, C., Lyons, J. A., Holl, R., Aragao, D., Arlow, D. H., Rasmussen, S. G., Choi, H. J., Devree, B. T. <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+irreversible+agonist-beta(2)+adrenoceptor+complex.">Structure and function of an irreversible agonist-beta(2) adrenoceptor complex.</a> Nature. 469, 236-240 (2011).</li><li>Shimamura, T., Shiroishi, M., Weyand, S., Tsujimoto, H., Winter, G., Katritch, V., Abagyan, R., Cherezov, V., Liu, W., Han, G. W., Kobayashi, T., Setvens, R. C., Iwata, S. <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+human+histamine+H1+receptor+complex+with+doxepin.">Structure of the human histamine H1 receptor complex with doxepin.</a> Nature. 475, 65-70 (2011).</li><li>Thompson, A. A., Liu, W., Chun, E., Katritch, V., We, H., Vardy, E., Huang, X. P., Trapella, C., Guerrini, R., Calo, G., Roth, B. L., Cherezov, V., Stevens, R. C. <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+nociceptin/orphanin+FQ+receptor+in+complex+with+a+peptide+mimetic.">Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic.</a> Nature. 485, 395-399 (2012).</li><li>Wu, B., Chien, E. Y., Mol, C. D., Fenalti, G., Liu, W., Katritch, V., Abagyan, R., Brooun, A., Wells, P., Bi, F. C., Hamel, D. J., Kuhn, P., Handel, T. M., Cherezov, V., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+CXCR4+Chemokine+GPCR+with+Small-Molecule+and+Cyclic+Peptide+Antagonists.">Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists.</a> Science. 330, 1066-1071 (2010).</li><li>Wu, H., Wacker, D., Mileni, M., Katritch, V., Han, G. W., Vardy, E., Liu, W., Thompson, A. A., Huang, X. P., Carroll, F. I. <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+human+kappa-opioid+receptor+in+complex+with+JDTic.">Structure of the human kappa-opioid receptor in complex with JDTic.</a> Nature. 485, 327-332 (2012).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-168 (2010).</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> J. Vis. Exp. , (2011).</li><li>Cherezov, V., 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=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., 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=Picolitre-scale+crystallization+of+membrane+proteins.">Picolitre-scale crystallization of membrane proteins.</a> J. Appl. Cryst. 39, 604-606 (2006).</li><li>W&#246;hri, A. B., Johansson, L. C., Wadsten-Hindrichsen, P., Wahlgren, W. Y., Fischer, G., Horsefield, R., Katona, G., Nyblom, M., Oberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Lipidic-Sponge+Phase+Screen+for+Membrane+Protein+Crystallization.">A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization.</a> Structure. 16, 1003-1009 (2008).</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=Rastering+strategy+for+screening+and+centring+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+microm+size+X-ray+synchrotron.">Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 microm size X-ray synchrotron.</a> 6, 587-597 (2009).</li><li>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=A+lipid's+eye+view+of+membrane+protein+crystallization+in+mesophases.">A lipid's eye view of membrane protein crystallization in mesophases.</a> Curr. Opin. Struct. Biol. 10, 486-497 (2000).</li><li>Briggs, J., Chung, H., 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=The+temperature-composition+phase+diagram+and+mesophase+structure+characterization+of+the+monoolein/water+system.">The temperature-composition phase diagram and mesophase structure characterization of the monoolein/water system.</a> J. Phys. Ii. 6, 723-751 (1996).</li><li>Qiu, H., 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=The+phase+diagram+of+the+monoolein/water+system:+metastability+and+equilibrium+aspects.">The phase diagram of the monoolein/water system: metastability and equilibrium aspects.</a> Biomaterials. 21, 223-234 (2000).</li><li>Misquitta, Y., Cherezov, V., Havas, F., Patterson, S., Mohan, J. M., Wells, A. J., Hart, D. J., 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=Rational+design+of+lipid+for+membrane+protein+crystallization.">Rational design of lipid for membrane protein crystallization.</a> J. Struct. Biol. 148, 169-175 (2004).</li><li>Caffrey, M., Porter, C. <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+for+Structure+Determination+using+Lipidic+Mesophases.">Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases.</a> J. Vis. Exp. , e1712 (2010).</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+mesophase.">Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophase.</a> J. Vis. Exp. , e4000 (2012).</li><li>Lyons, J. A., Aragao, D., Slattery, O., Pisliakov, A. V., Soulimane, T., 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=Structure+insights+into+electron+transfer+in+caa(3)-type+cytochrome+oxidase.">Structure insights into electron transfer in caa(3)-type cytochrome oxidase.</a> Nature. 10, (2012).</li></ol>

الإعلان عن أي تضارب في المصالح.

في هذه المقالة الفيديو أثبتنا كيف يتم حصاد بلورات نمت في mesophase شحمي والبرد تبريد استعدادا لاستخدامها في جمع البيانات والحيود في نهاية المطاف لتحديد الهيكل. يمكن للmesophase استضافة تكون المرحلة لزج ودبق أو مكعب المرحلة الإسفنج أكثر مرونة 4. كيف يتم فتح الزجاج لوحات ساندويتش وكيف يتم حصاد هذه البلورات كثيرا يعتمد على نوع mesophase. ولذا فمن المهم أن تعرف أي واحد اثنين من يتعامل مع في وقت مبكر. هوية الدهون استضافة ومرسب المستخدمة هي هامة في هذا الصدد، والمظهر الخارجي للبلعة mesophase في التبلور بشكل جيد يمكن استخدامها لتمييزها عن بعضها البعض (الشكل 3). وقد تجلى الحصاد من كل أنواع mesophase في هذه المقالة. حصاد بلورات صغيرة من mesophase شحمي في لوحات ساندويتش الزجاج هو عملية مضنية تتطلب وقتا ومهارة، experيتزوج والصبر ومطرد من ناحية. من المهم أن يوضع جانبا مبلغ مناسب من الوقت لجمع وإعداد المختبر حتى يتسنى لجميع اللوازم والمعدات هي في متناول اليد مقدما. A الشخص الثاني للمساعدة في الحصاد ليست ضرورية ولكنها أوصت و. يمكن أن تساعد الشخص مع توريد لوحات ما قبل ملحوظ على الفرد القيام الحصاد وكذلك وضع البرد المبردة الحلقات مزودة بلورات تحصد في عفريت التخزين. يمكن للمساعد أيضا أن تلعب دورا هاما في دعم توثيق الملاحظات على بلورات التي قدمت خلال الحصاد التي قد تكون حاسمة خلال جمع البيانات حيود. في حالة عدم وجود مساعد، يمكن أن تستخدم صوت تنشيط السمعية وتسجيل الجهاز لميزة للتوثيق. وبعد بروتوكول الموصوفة في هذه المقالة مساعدة في الحصول على عارض وتشغيلها مع الحصاد وضوح الشمس. ومع ذلك، فمن المهم أن نقدر أن العملية ليست واضحة وأن تحليل مخاطر الآفاتوهناك حاجة إلى إطلاق ctice قبل الحصاد قيمة بلورات البروتين الغشاء. ولذلك فمن المستحسن أن جربت لوحات اختبار مع بلورات من البروتينات التي لا قيمة خاصة مع الأول. وهذا سيوفر على المبتديء مع خبرة قيمة في قطع الزجاج، وإزالة شظايا الزجاج، ورفع حتى ساترة من خلال mesophase، وذلك باستخدام ميزة الاستقطاب على المجهر لرؤية بلورات وتعقبهم أثناء الحصاد، وأخيرا التعامل مع أنواع مختلفة من mesophases والحصاد منها. نسيج من mesophase، وبالتالي السهولة التي يمكن حصادها بلورات، هل تغير مع مرور الوقت خلال التبلور. ولذلك فمن المهم لممارسة الحصاد مع بلورات أقل قيمة ولكن مع تلك التي نمت في ظل نفس الظروف ومنها أكثر قيمة. فمن الممكن أن تنمو بلورات الليزوزيم وthaumatin من قبل في المتوسط ​​شحمي أو أسلوب المرحلة مكعب 28 و ينبغي consid هذهered عن طريق كسب الألفة مع المواد والأسلوب. ينبغي للمرء أن ينظر أيضا العمل مع البروتين خالية mesophase أول من تعلم من تقلبات والخمسين. تم الانتهاء من جميع الإجراءات أثبتت هنا في C ° 20 مريحة أو ما يقرب من ذلك. فمن الممكن أن تنمو بلورات من قبل في طريقة المتوسط ​​في درجات حرارة منخفضة. وبالتالي، يمكن استخدام الدهون monoolein كما في حالة استضافة المرحلة متبدل الاستقرار في 4 1،2،29،30 C °. والبديل هو استخدام تصميم بعقلانية 7،9 MAG لبلورة درجة حرارة منخفضة 31. نفعل انخفاض درجة الحرارة بشكل روتيني crysallogenesis مع بعض الأهداف البروتين الغشاء. في هذه الحالة، يتم نمو البلورات والحصاد في الثلاجة المشي في ° C 4. العمل في ظل هذه الظروف والتحديات الخاصة به وليس أقلها الذي هو الحاجة إلى الملابس دافئة ومريحة. الخطوة التالية في العملية الشاملة لتصميم هيكل الجزيئات باستخدام بلورةtallography هو جمع البيانات حيود على بلورات حصاد وبرد الإضافية كما هو موضح في هذه المقالة. في المتوسط ​​نابعة من بلورات وعادة ما تكون صغيرة. ومع ذلك، فقد كانت مفيدة لجمع البيانات حيود ممكن مع وجود بلورات بعدا أقصى 20 ميكرون 9. لهذا الغرض، يتم استخدام شعاع السنكروترون الصغيرة X-الإشعاع وهو محور مقالة منفصلة إن الرب في هذه السلسلة 32،33.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> مكونات </td></tr><tr><td> منحني ملاقط </td><td> سيجما </td><td> F4142 </td><td> أداة </td></tr><tr><td> نصائح ماصة المتاح </td><td> جيلسون </td><td> مختلف </td><td> يمكن التخلص منها </td></tr><tr><td> رغوة ديوار </td><td> Spearlab </td><td> FD-500 </td><td> أداة </td></tr><tr><td> حاويات النفايات الزجاج والمعادن </td><td> دانيلز الرعاية الصحية </td><td> DD479OL </td><td> أداة </td></tr><tr><td> حصاد الحلقات </td><td> MiTeGen </td><td> مختلف </td><td> أداة </td></tr><tr><td> حصاد المجهر </td><td> نيكون </td><td> SMZ1500 </td><td> أداة </td></tr><tr><td> مختبر الكمبيوتر المحمول </td><td> مختلف </td><td> NA </td><td> أداة </td></tr><tr><td> دفع المغناطيسيعصا زر التحميل عينة </td><td> البحث هامبتون / الأبعاد الجزيئية </td><td> HR4-729/MD7-411 </td><td> أداة </td></tr><tr><td> كرة الصولجان الأصلي (للاستخدام مع طراز ALS الروبوتات فقط) </td><td> نظم تحديد المواقع الكريستال </td><td> CP-111-035 </td><td> أداة </td></tr><tr><td> Pipetting الأجهزة </td><td> جيلسون </td><td> مختلف </td><td> أداة </td></tr><tr><td> الكثيرمن الحلول </td><td> مختلف </td><td> مختلف </td><td> كاشف </td></tr><tr><td> عفريت بنت البرد تونغ </td><td> نظم تحديد المواقع الكريستال </td><td> CP-111-030 </td><td> أداة </td></tr><tr><td> عفريت قصب شحن الرف (ALS الأصلي على غرار) مع مقبض قضيب مدمن مخدرات وتأمين </td><td> نظم تحديد المواقع الكريستال </td><td> CP-111-029 </td><td> أداة </td></tr><tr><td> تنقية المياه </td><td> ميليبور </td><td></td><td> كاشف </td></tr><tr><td> نظارات السلامة </td><tد&gt; متنوع </td><td> NA </td><td> أداة </td></tr><tr><td> أسس دبوس عينة - المغناطيسي (غير النحاس) </td><td> نظم تحديد المواقع الكريستال </td><td> CP-111-015 </td><td> أداة </td></tr><tr><td> الشحن ديوار </td><td> تايلور وارتون </td><td> CX100 </td><td> أداة </td></tr><tr><td> الأنسجة </td><td> NA </td><td> NA </td><td> يمكن التخلص منها </td></tr><tr><td> التنغستن وكربيد قطع الزجاج (الخطاط TCT) </td><td> عليوي أدوات (يوفيل، UK) </td><td> 633657 </td><td> أداة </td></tr></tbody></table>

harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography

طريقا هاما لفهم كيفية البروتينات تعمل على مستوى الآلية هو أن يكون هيكل البروتين المستهدف المتاحة، من الناحية المثالية في قرار الذرية. في الوقت الحاضر، لا يوجد سوى طريقة واحدة لالتقاط هذه المعلومات كما ينطبق على البروتينات الغشاء لا يتجزأ (الشكل 1)، وأنها تشكل المجمعات، وهذا الأسلوب هو الجزيئات البلورات بالأشعة السينية (MX). للقيام البلورات هناك حاجة MX نوعية حيود التي، في حالة من البروتينات الغشاء، لا تشكل بسهولة. وقد اكتسب طريقة لبلورة البروتينات الغشاء الذي ينطوي على استخدام mesophases شحمي، وتحديدا في المراحل مكعب والاسفنج 1-5، اهتماما كبيرا من المقرر في وقت متأخر إلى النجاحات وكان له في مستقبلات البروتين G مجال يقترن 6-21 ( <a href="http://www.mpdb.tcd.ie" target="_blank">شبكة الاتصالات العالمية . mpdb.tcd.ie</a> ). ومع ذلك، وهي طريقة، المشار إليه فيما لفي المتوسط ​​شحمي أو أسلوب المرحلة مكعب، ويأتي مع التقنية الخاصة بهاالتحديات. هذه تنشأ، في جزء منه، نظرا لطبيعة لزجة لزجة وعموما من mesophase شحمي فيه البلورات، والتي غالبا ما تكون بلورات متناهية الصغر، وتنمو. بلورات يصبح من الصعب التلاعب نتيجة لذلك وخاصة أثناء الحصاد 22،23. تنشأ مشاكل أيضا في الخطوة التي تسبق الحصاد الذي يتطلب أن يتم فتح الزجاج لوحات ساندويتش التي تنمو بلورات (الشكل 2) 24،25 لفضح البلعة mesophase، وبلورات فيه، للحصاد، البرد التبريد وX في نهاية المطاف حيود أشعة جمع البيانات. المتغيرات mesophase مكعب والإسفنج (الشكل 3) من الذي يجب أن يكون حصاد بلورات مختلفة rheologies عميقا 4،26. المرحلة مكعب هو لزج ودبق أقرب إلى معجون الأسنان سميكة. على النقيض من ذلك، فإن المرحلة الاسفنج هو أكثر مرونة مع وجود اتجاه متميز في التدفق. وفقا لذلك، ونهج مختلفة لبلورة فتح الآبار containiوتسمى بلورات نانوغرام نموا في المرحلة مكعب والاسفنج لكما في الواقع هناك حاجة لأساليب مختلفة من بلورات حصاد أنواع mesophase اثنين. بروتوكولات لتفعل ذلك تم تنقيحها وتنفيذها في الغشاء الأحياء الهيكلية والوظيفية (MS &amp; FB) مجموعة، وصفها بالتفصيل في هذه المقالة إن الرب (الشكل 4). يتم إعطاء أمثلة على الحالات التي تحصد بنجاح بلورات البرد ويبرد. ونحن نقدم أيضا أمثلة على الحالات التي تنشأ مشاكل التي تؤدي إلى خسارة لا تعوض من بلورات وتصف كيف يمكن تجنب هذه المشاكل. في هذه المقالة يتم توفير العارض مع خطوة بخطوة تعليمات لفتح الآبار الزجاج تبلور ساندويتش، للحصاد والبرد التبريد بلورات من البروتينات الغشاء نموا في مكعب وعلى مراحل الاسفنج.

Watch this Scientific Journal Video about Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography at JoVE.co

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

يوصف هنا الإجراءات المنفذة في الغشاء Caffrey الهيكلية والوظيفية لمجموعة الأحياء بلورات الحصاد والبرد بارد من البروتين غشاء شحمي تزرع في مراحل مكعب والإسفنج لاستخدامه في تحديد هيكل الجزيئات باستخدام الأشعة السينية البلورات.

الحصاد والبرد تبريد بلورات من بروتينات الغشاء يزرع في Mesophases شحمي لتحديد هيكل للعلم البلورات ضخم الجزيئات

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

harvesting-cryo-cooling-crystals-membrane-proteins-grown-lipidic

Research

JoVE Journal

Biology

24.7K Views.  Trinity College Dublin. يوصف هنا الإجراءات المنفذة في الغشاء Caffrey الهيكلية والوظيفية لمجموعة الأحياء بلورات الحصاد والبرد بارد من البروتين غشاء شحمي تزرع في مراحل مكعب والإسفنج لاستخدامه في تحديد هيكل الجزيئات باستخدام الأشعة السينية البلورات.

Video: Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

بلورة البروتينات الغشائية لتحديد الهيكل باستخدام Mesophases Lipidic

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

على أهمية تركيز البروتين الصحيح لحركية وتقارب في تحديد هيكل وظيفة التحليل

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

تقييم المحاكمات تبلور ثنائي الأبعاد للبروتينات غشاء الصغيرة للدراسات البيولوجيا الهيكلية التي البلوريات الكترون

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

بلورة البروتينات في الغشاء Mesophases Lipidic

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

الإنتاجية العالية للبروتينات غشاء البلورة باستخدام أسلوب Bicelle Lipidic

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

استخدام روبوت لارتفاع الإنتاجية البلورة من بروتينات الغشاء في Mesophases شحمي

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

حيوي داخلي المجهري للهياكل التصوير التحت خلوية في الفئران لايف تعرب عن البروتينات الفلورية

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

مناعي للبروتينات الغشاء الخارجي بواسطة التدفق الخلوي من الميتوكوندريا المعزولة

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.