ישנם רב שתרמו לעבודה זו ורובם מן הקרום המבני קפריי וקבוצת ביולוגיה פונקציונלית, שניהם חברים בעבר ובהווה. לכל, ובמיוחד לJingquan טאן וג&#39;וזף ליונס, אנו מרחיבים את התודה וההערכה החמה שלנו. עבודה זו נתמכה בחלקו על ידי מענקים מקרן המדע אירלנד (07/IN.1/B1836), המכונים הלאומיים לבריאות (GM75915, P50GM073210 וU54GM094599), ועלות FP7 ופעולות Curie מארי (CM0902 וPIEF-GA-2009 -235612).

<ol>
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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>

אין ניגודי האינטרסים הכריזו.

במאמר זה וידאו שהראינו כיצד גבישים גודלו בmesophase lipidic נקצרים וcryo התקררו בתכשיר לשימוש באיסוף נתונים עקיפים וסופו של דבר לקביעת מבנה. Mesophase האירוח יכול להיות שלב מעוקב הצמיג ודביק או שלב הספוג יותר נוזלי 4. איך את צלחות כריכי הזכוכית נפתחות ואיך את הגבישים נקצרים מאוד תלוי בסוג mesophase. לכן חשוב לדעת איזו משני אחד לא עוסק בבעוד מועד. זהותו של שומני האירוח והמזרז בשימוש הן חשובה בהקשר זה, ואת המראה הפיזי של בולוס mesophase בגיבוש גם יכול לשמש כדי להבדיל ביניהם (איור 3). מסיק משני סוגי mesophase הומחש במאמר זה. קציר גבישים קטנים מmesophase lipidic בצלחות זכוכית כריך הוא תהליך קפדני שדורש זמן, מיומנות, experience, סבלנות ויד יציבה. חשוב להניח בצד את הכמות מתאימה של זמן לקצירה ולהקים את המעבדה כך שכל האספקה ​​והציוד הם בהישג יד מראש. אדם שני, שיסייע בקצירה אינו הכרחי אך מומלץ. אותו האדם יכול לעזור באספקת לוחות מסומנים מראש לאדם עושה קצירה, כמו גם הצבת לולאות רכובות cryo מקוררים עם גבישים נקטפו לתוך דיסקוס האחסון. העוזר יכול גם לשחק תפקיד חשוב בתמיכה בתיעוד תצפיות על הגבישים בוצעו במהלך הקצירה שעשויה להוכיח מכריע במהלך איסוף נתוני עקיפה. בהעדרו של עוזר, מכשיר קול המופעל שמע הקלטה יכול לשמש יתרון לתיעוד. בעקבות הפרוטוקול המתואר במאמר זה יעזור לי המציג והפעלה עם קצירת גביש. עם זאת, חשוב להבין, שהתהליך אינו פשוט וכי practice נחוץ לפני ההשקה לקצירת גבישי חלבון קרום יקרים ערך. לכן מומלץ שצלחות בדיקה עם גבישים של חלבונים שאינם יקרים במיוחד שהתנסו בראשון. זה יספק את החניך עם ניסיון רב ערך בחיתוך זכוכית, הסרת רסיסי זכוכית, מרים coverglass מרחבי mesophase, תוך שימוש בתכונת הקיטוב במיקרוסקופ כדי לראות גבישים ולעקוב אחריהם במהלך קציר, ולבסוף טיפול בסוגים השונים של mesophases וקציר מהם. המרקם של mesophase, ובהרחבה את הקלות שבה ניתן לקצור גבישים, משתנה עם זמן במהלך התגבשות. לכן חשוב לתרגל קצירה עם גבישים פחות יקרים אבל עם אלה שגדלו באותם תנאים כמו אלה יקרים יותר. זה אפשרי לגדל גבישים של Lysozyme וthaumatin ידי בטווה או שיטת שלב מעוקב lipidic 28 ואלה צריכים להיחשב כנעלי ספורט בדרך של צובר היכרות עם חומרים ואת השיטה. כדאי גם לשקול לעבוד עם mesophase חלבון החופשי ראשון ללמוד מהגחמות שלו. הנהלים הפגינו כאן היו הכל נעשה ב20 נוח ° C או בסמוך לכך. זה אפשרי לגדל גבישים ידי בשיטה טווה בטמפרטורות נמוכות יותר. לפיכך, monoolein כשומני אירוח במדינת שלב metastable ניתן להשתמש ב 4 ° C 1,2,29,30. אלטרנטיבה היא להשתמש 7.9 MAG תוכנן באופן רציונלי להתגבשות טמפרטורה נמוכה 31. אנחנו עושים crysallogenesis טמפרטורה נמוכה באופן שגרתי עם מטרות חלבון קרום מסוימות. במקרה זה, גידול גביש והקציר נעשה בהליכה, ב4 ° C מקרר. עובד בתנאים כאלה, יש אתגרים משלו לא פחות מזה הוא הצורך בלבוש חם ונוח. השלב הבא בתהליך הכולל של קביעת מבנה באמצעות crys macromoleculartallography הוא לאסוף נתונים עקיפים על גבישים נקטפו וSnap-התקרר כפי שמודגם במאמר זה. במבוגר טווה גבישים הם בדרך כלל קטנים. עם זאת, איסוף נתונים עקיפים שימושי היה אפשרי בגבישים בעלי ממד מקסימום של 20 מיקרומטר 9. לצורך כך, מייקרו קרן ה-X קרינת סינכרוטרון משמשת ונמצאת במוקד המאמר יופיטר נפרד בסדרת 32,33 זה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> רכיבים </td></tr><tr><td> פינצטה מעוקלת </td><td> סיגמא </td><td> F4142 </td><td> כלי </td></tr><tr><td> טיפי פיפטה חד פעמיים </td><td> Gilson </td><td> שונה </td><td> חד פעמי </td></tr><tr><td> הקצף דיואר </td><td> Spearlab </td><td> FD-500 </td><td> כלי </td></tr><tr><td> מכולות פסולות זכוכית ומתכת </td><td> דניאלס בריאות </td><td> DD479OL </td><td> כלי </td></tr><tr><td> לולאות קצירה </td><td> MiTeGen </td><td> שונה </td><td> כלי </td></tr><tr><td> קציר מיקרוסקופ </td><td> ניקון </td><td> SMZ1500 </td><td> כלי </td></tr><tr><td> מחברת מעבדה </td><td> שונה </td><td> NA </td><td> כלי </td></tr><tr><td> דחיפה מגנטיתמדגם שרביט טעינת כפתור </td><td> המפטון מחקר / ממדים מולקולריים </td><td> HR4-729/MD7-411 </td><td> כלי </td></tr><tr><td> פאק המקורי (לשימוש עם רובוטים מחא בסגנון בלבד) </td><td> מערכות מיקום Crystal </td><td> CP-111-035 </td><td> כלי </td></tr><tr><td> מכשירי pipetting </td><td> Gilson </td><td> שונה </td><td> כלי </td></tr><tr><td> פתרונות נחפזים </td><td> שונה </td><td> שונה </td><td> מגיב </td></tr><tr><td> פאק בנט Cryo-טונג </td><td> מערכות מיקום Crystal </td><td> CP-111-030 </td><td> כלי </td></tr><tr><td> קני פאק גנזו משלוח (ALS המקורי בסגנון) עם ידית מעוקלת ומוט נעילה </td><td> מערכות מיקום Crystal </td><td> CP-111-029 </td><td> כלי </td></tr><tr><td> מים מטוהרים </td><td> Millipore </td><td></td><td> מגיב </td></tr><tr><td> משקפי בטיחות </td><tד&gt; אמנים שונים </td><td> NA </td><td> כלי </td></tr><tr><td> בסיסי פין דוגמה - מגנטי (ללא נחושת) </td><td> מערכות מיקום Crystal </td><td> CP-111-015 </td><td> כלי </td></tr><tr><td> המשלוח דיואר </td><td> טיילור-ורטון </td><td> CX100 </td><td> כלי </td></tr><tr><td> רקמות </td><td> NA </td><td> NA </td><td> חד פעמי </td></tr><tr><td> טונגסטן קרביד-חותך זכוכית (כותב TCT) </td><td> הגדרות Silverline (Yeovil, בריטניה) </td><td> 633657 </td><td> כלי </td></tr></tbody></table>

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

מסלול חשוב להבנה כיצד חלבונים לתפקד ברמה מכניסטית הוא לקבל את המבנה של חלבון המטרה הזמין, באופן אידיאלי ברזולוציה אטומית. נכון להיום, יש רק דרך אחת כדי ללכוד מידע מסוג זה כפי שהוחל על חלבונים אינטגרליים בממברנה (איור 1), ואת הקומפלקסים שהם יוצרים, ושיטה שהיא רנטגן קריסטלוגרפיה macromolecular (MX). כדי לעשות גבישים באיכות עקיפה מושלמת יש צורך בו, במקרה של חלבונים בממברנה, לא יוצר בקלות. שיטה לגיבוש חלבונים בממברנה, הכרוכים בשימוש בmesophases lipidic, במיוחד את השלבים העוקבים וספוג 1-5, זכתה לתשומת לב רבה של עקב האיחור להצלחות שהיו לה בתחום הקולטן G-חלבון בשילוב 6-21 <a href="http://www.mpdb.tcd.ie" target="_blank">(www . Mpdb.tcd.ie).</a> עם זאת, השיטה, יקרא להלן בטווה או שיטת שלב מעוקב lipidic, מגיע עם טכני שלואתגרים. אלה נובעים, בחלקו, בשל האופי בדרך הצמיג ודביק של mesophase lipidic שבו הגבישים, אשר לעתים גבישי מייקרו, לגדול. מניפולצית גבישים הופכת להיות קשה כתוצאה ובעיקר במהלך קצירת 22,23. בעיות מתעוררות גם בשלב שלפני קציר אשר דורש כי צלחות זכוכית הכריך שבו גדלים הגבישים (איור 2) 24,25 נפתחות כדי לחשוף את בולוס mesophase, ובו הגבישים, לקטיף, קירור וcryo-X סופו של דבר איסוף נתוני עקיפה-ray. וריאנטים mesophase מעוקבים וספוג (איור 3) שממנה יש לי rheologies נקטפו גבישים שונים השונה המהותי 4,26. השלב מעוקב הוא צמיג ודביק דומה למשחת שיניים עבה. לעומת זאת, בשלב הספוג הוא יותר נוזל עם נטייה מובהקת לזרימה. בהתאם לכך, גישות שונות להתגבשות פתיחת הבארות containiגבישי ng גדלו במעוקב ושלב הספוג נקראים לכפי שאכן שיטות שונות נדרשות לקציר גבישים משני סוגי mesophase. פרוטוקולים לעושים בדיוק את זה כבר מעודן ומיושם בממברנה ביולוגיה מבנית ותפקודית (MS &amp; FB) קבוצה, והם מתוארים בפירוט במאמר זה יופיטר (איור 4). דוגמאות לכך הן נתונה של מצבים שבם מסתיימים בהצלחת הגבישים וcryo התקררו. אנו מספקים גם דוגמאות למקרים בם מתעוררות בעיות שהובילו לאובדן בלתי ההפיך של גבישים ונתאר כיצד בעיות אלה ניתן למנוע. במאמר זה מציג מסופק עם הוראות שלב אחר שלב לפתיחת בארות התגבשות כריך זכוכית, לקציר ולגבישי cryo-קירור של חלבוני קרום גדלו במעוקב ובשלבים ספוגים.

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

במסמך מתואר נהלים המיושמים בממברנה קבוצת ביולוגיה המבנית ופונקציונלית לגבישי חלבון קרום קציר וcryo-מגניב שגודלו בשלבים עוקבים וספוג lipidic לשימוש בקביעת מבנה באמצעות קריסטלוגרפיה macromolecular רנטגן קאפרי.

קצירה וCryo-קירור גבישים של חלבונים בממברנה גדלו בMesophases Lipidic לקביעת מבנה על ידי קריסטלוגרפיה Macromolecular

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

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Research

JoVE Journal

Biology

24.7K Views.  Trinity College Dublin. במסמך מתואר נהלים המיושמים בממברנה קבוצת ביולוגיה המבנית ופונקציונלית לגבישי חלבון קרום קציר וcryo-מגניב שגודלו בשלבים עוקבים וספוג lipidic לשימוש בקביעת מבנה באמצעות קריסטלוגרפיה macromolecular רנטגן קאפרי.

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.

גיבוש חלבונים בממברנה עבור קביעת מבנה באמצעות 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.

חשיבותה של ריכוז חלבון נכון עבור קינטיקס וקביעת זיקה בניתוח מבנה פונקציה

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.

הערכת דו מימדי ניסויים התגבשות של חלבונים בממברנה קטנים ללימודי לביולוגיה מבנית על ידי קריסטלוגרפיה אלקטרונים

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.

התגבשות של חלבונים בממברנה ב 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.

תפוקה גבוהה התגבשות של חלבונים בממברנה שימוש בשיטה Lipidic Bicelle

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.

שימוש ברובוט להתגבשות תפוקה גבוהה של חלבונים בממברנה בMesophases Lipidic

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.

מיקרוסקופית Intravital למבני ההדמיה subcellular בעכברים חיים לבטא חלבוני פלורסנט

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.

Immunodetection של החלבונים בממברנה חיצוניים על ידי cytometry הזרימה של מיטוכונדריה מבודדת

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.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</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.