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Detergent-protein and detergent-lipid interactions : implications for two-dimensional crystallization of membrane proteins and development of tools for high throughput crystallography

机译:洗涤剂 - 蛋白质和去污剂 - 脂质相互作用:对膜蛋白质的二维结晶的影响和用于高通量晶体学的工具的开发

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摘要

2.1 Scope of this ThesisudThis thesis represents an attempt to enlighten the role of the detergent in reconstitution and more specificallyudin two-dimensional (2D) crystallogenesis of membrane proteins. The construction of a tool for preciseudand routine measurements of detergent concentrations provided a valuable tool for better understandingudand controlling the detergent issue. Additionally, a novel approach for detergent removal in 2D crystallization,udi.e. the use of cyclodextrins was explored and a nanoliter dispensing high throughput tool wasuddeveloped allowing for profound and sophisticated screening of optimal conditions for protein reconstitutionudand crystallization.udud2.2 Combining Electron Microscopyudand Atomic ForceudMicroscopyudAlthough electron crystallography has proven to beuda powerful approach to structure determination ofudmembrane proteins (for a recent example see (Gonenudet al., 2005)) successes are somehow restrictedudto certain classes of membrane proteins (e.g., outerudmembrane porins, aquaporins, naturally occurringudcrystalline proteins). This is mainly due to the stabilityudof these proteins with respect to biochemicaludmanipulation. One can not exclude however, thatudthese are simply more amenable to crystallizationuddue to the nature of their molecular surfaces.ud2D crystallization exhibits several advantagesudcompared to 3D crystallization of membrane proteins:udThe simple fact that the proteins are allowedudto reside in a native-like environment, i.e.,udthe membrane and that their function is not impairedudby the lateral crystal contacts is of considerableudinterest. If structural investigations shall notudbe restricted to static snapshots of different conformationsudand moreover structure-function relationshipsudshall be established, then electron microscopyud(EM) in combination with atomic force microscopyud(AFM) surely represent a valuable approach.udIn Chapter 2 the combination of such data hasudbeen successfully applied to the ammonium transporterudAmtB from Escherichia coli. The aim was touddetermine the crystal packing of the double-layeredud2D crystals of AmtB by AFM in order to process theudcryo EM data. Additionally, the AFM images, dueudto their outstanding signal-to-noise ratio, enabledudthe direct visualization of trimers in the reconstitutedudmembranes. The topographical data fromudthe AFM allowed the assessment of a single layerudwithin the double layered crystals.ud2.3 Investigating the Role of theudDetergentudIn Chapter 3 the development of a fast and preciseudmethod for detergent concentration determinationudis presented. The robustness and wide applicationudrange of this method has been demonstratedudby comparing concentrations of radioactivelyudlabeled dodecyl-[beta],D-maltoside (DDM) withudmeasured contact angles, by measuring the amountudof DDM bound to the proton/galactose symporterudGalP from E. coli, by measuring the effectsudof 100 mM NaCl on the cmc of dodecyl-N,Ndimethylamine-udN-oxide, by characterizing the surfaceudenergy of Parafilm, and finally by revealingudthe stoichiometry of complex formation betweenudmethyl-[beta]-cyclodextrin (MBCD) and different de-ududtergents. The possibility of performing such measurementsudroutinely in membrane biochemistry isudunique compared to all other methods available touddate.udChapter 4 addresses the major aspects of detergentuduse in membrane protein purification andudcrystallization. First, the stability of GalP in differentuddetergents is assessed, unveiling profounduddifferences in the capacity of detergents to keepudthe protein in solution. Second, it is demonstrated,udthat the amount of a detergent, i.e., dodecyl-�,Dmaltoside,udbound to a protein can be controlledudduring purification. At last the amount of differentuddetergents for solubilization of E. coli lipids isuddetermined, showing differences in the mechanismsudby which detergents promote solubilization.udBanerjee et al. (Banerjee et al., 1995) examinedudthe preferential affinity of detergents for differentudlipids in mixed membranes (such as biological membranes).udThey showed that different detergents extractudthe serotonin 5-HT1A receptor from nativeudmembranes along with different lipids. The effectudis considerable and might explain why different detergentsudexhibit such a different ability to keep audprotein in its native state, because some might simplyudnot be able to co-solubilize native lipids essentialudfor the stability (and function) of the protein.udThe amount of detergent bound to a protein isudof special interest when using dialysis or dilutionudfor detergent removal. Furthermore, in most casesudthe protein must not be exposed to excess detergentudwhich anyway fails to satisfactorily mimic theudnative bilayer. As pointed out in the discussion ofudChapter 4, protein reconstitution is facilitated whenudthe detergent collar that is present around the hydrophobicudregion of membrane proteins in solutionudis near its solubility limit (Psol).udThe same is true for the lipid: Reconstitution isudlikely to happen when liposomes are forming, thereforeudan excess of detergent is not desirable either.udAdditionally, even detergents known to have adverseudeffects on protein stability can be used forudlipid solubilization, given that they are present at audminimal concentration. The use of detergent mixturesudin crystallization can also have the effect ofudreducing the size of the detergent collar around theudprotein. Moreover, the free detergent concentrationudin detergent mixtures is altered by the presence ofudthe second species and can be crucial to the formationudof crystals in some cases (Koning, 2003).udWhen using minimal amounts of detergent in audcrystallization mixture, special care should be takenudwith respect to the formation of ternary micelles.udIdeally, equilibration of the ternary mixtures priorudto detergent removal needs to be completed.ud2.4 The Use of Cyclodextrins forudHigh Thorughput 2D Crystallizationudof Membrane ProteinsudChapter 5 demonstrates the feasibility of theudcyclodextrin-based detergent removal for twodimensionaludcrystallization. The possibility ofudchoosing different kinetics, simply by adding differentudamounts of cyclodextrin at various time intervalsudis one of the major advantages of this method.udBy implementing optical spectroscopy, it would beudpossible to slow down the detergent removal rate atudthe onset of proteoliposome and 2D crystal formation.udAs pointed out by Lichtenberg et al. (Lichtenbergudet al., 2000) the rate of detergent removal hasudto be slow enough to allow for detergent-inducedudvesicle size growth, a process which is usually quiteudslow. This aspect is important to keep in mind asudone defines the rate of detergent neutralization (inudcontrast to dialysis). At a first glance one mightudthink that in this respect the cyclodextrin approachudbears no advantage compared to dialysis. However,udthe rate of low-cmc detergent removal using dialysisudcan be too slow, thereby keeping the protein outudfrom its native environment for too long, ultimatelyudpromoting its precipitation.udIn Chapter 6 we present an apparatus for paralleludquantitative reconstitution and 2D crystallizationudof membrane proteins. Cyclodextrin provides audunique opportunity for high throughput implementationudcompared to other methods available today.udProtein concentrating through controlled evaporationudwith concomitant detergent neutralization (toudprevent detergent concentrating) is advantageousudcompared to commercially available protein concentratinguddevices which very often concentrate detergentudmicelles too. Moreover, the possibility of usingudone protein preparation for wide screening ensuresudthat inconsistencies in results arising from preparativeuddifferences are excluded. Often, the detergentudand lipid concentration of the purified protein areudill characterized, and this variability may be a causeudfor much of the irreproducibility and failure in crystallizationud(Wiener, 2004).udSo far the use of wide screening matrices (sparseudmatrix design) in 2D crystallography was restrictedudby the enormous number of experiments andududamount of protein needed for a rigorous screening.udThe presented machine makes it possible to partiallyudcompensate for the first bottleneck in proteinudstructure elucidation, which is the over-expressionudof membrane proteins.udFig. 2.1 summarizes the screening strategy basedudon the criteria discussed in Chapter 6 and above.udScreening efficiency is provided by the subdivisionudof the problem into multiple subproblems and byudtheir sequential screening.udWith the high throughput approach however, audnew bottleneck arises as one will produce a largeudnumber of crystallization trials, which have to beudscreened for their outcome. Therefore –in analogyudto the x-ray community– the development of automatedudsample preparation and automated electronudmicroscopic analysis would provide substantial supportudto the 2D crystallographer.udCombining step-by-step identification of key valuesudnecessary for crystallization (and/or efficient reconstitution)udtogether with high throughput screeningudmatrices opens up new prospects in the enududdeavor to membrane protein structure and functionuddetermination. Now it is possible to apply audsemi-rational screening strategy and this might contributeudto transform 2D crystallization from art toudscience (Jap et al., 1992).
机译:2.1本论文的范围本论文代表了一种尝试,以启发去污剂在重组过程中的作用,更具体地说,是在膜蛋白的二维(2D)结晶过程中发挥作用。用于精确/常规测量洗涤剂浓度的工具的构造为更好地理解/理解洗涤剂问题提供了有价值的工具。此外,还有一种用于2D结晶去除洗涤剂的新颖方法。探索了使用环糊精的方法,并开发了纳升分配高通量工具,可以对蛋白质重构 udand结晶的最佳条件进行深刻而复杂的筛选。 ud ud2.2结合电子显微镜 ud和原子力 udMicroscopy ud尽管电子晶体学已被证明是 ud膜蛋白结构测定的强大方法(有关最新示例,请参见(Gonen udet等人,2005)),成功以某种方式限制了 ud膜的某些类型的膜蛋白(例如,外层 udpor膜孔蛋白) ,水通道蛋白,天然超结晶蛋白)。这主要是由于这些蛋白质相对于生化/操纵的稳定性。但是,不能排除的是,由于分子表面的性质,这些化合物更易于结晶。 ud2D结晶具有与膜蛋白的3D结晶相比更多的优势: ud允许蛋白质的简单事实 udto驻留在一个类似自然的环境中,即 u膜,并且其功能不会因横向晶体接触而受到损害 udinterest。如果结构研究不应该局限于不同构象的静态快照 ud,而且应该建立结构-功能关系 ud,那么电子显微镜 ud(EM)结合原子力显微镜 ud(AFM)无疑是一种有价值的方法在第二章中,这些数据的组合已成功应用于来自大肠杆菌的铵转运蛋白udAmtB。目的是通过AFM确定AmtB的双层ud2D晶体的晶体堆积,以便处理udcryo EM数据。此外,由于其出色的信噪比,AFM图像可以使重组后的三聚氰胺中的三聚体直接可视化。 AFM的地形数据允许对双层晶体中的单层 ud进行评估。 ud2.3研究 udDetergent ud的作用在第3章中,开发了一种快速,精确的 udmethod用于测定洗涤剂浓度迪迪斯介绍。通过比较放射性未标记的十二烷基-β,D-麦芽糖苷(DDM)的浓度和未测量的接触角,通过测量与质子结合的DDM的量 ud,证明了该方法的鲁棒性和广泛的应用范围。 /半乳糖同向转运蛋白 udGalP来自大肠杆菌,通过测量100 mM NaCl对十二烷基-N,N-二甲胺-udN-氧化物的cmc的影响,通过表征对位膜的表面 udenergy,最后通过揭示 ud化学计量甲基-β-环糊精(MBCD)与不同去污剂之间的复合物的形成。与迄今为止所有可用的其他方法相比,在膜生物化学中常规地进行此类测量的可能性是不寻常的。第四章探讨了膜蛋白纯化和结晶中去垢剂的主要方面。首先,评估了GalP在不同洗涤剂中的稳定性,揭示了洗涤剂将蛋白质保留在溶液中的能力的深刻差异。其次,证明了在纯化过程中,可以控制蛋白质上所附着的去污剂(即十二烷基-麦芽糖苷)的量。最后,确定了用于溶解大肠杆菌脂质的不同洗涤剂的数量,这表明洗涤剂促进溶解的机理不同。 udBanerjee等人。 (Banerjee et al。,1995)研究了洗涤剂对混合膜(例如生物膜)中不同脂质的优先亲和力。 ud他们表明,不同的洗涤剂从天然膜中提取 5-羟色胺5-HT1A受体以及不同的脂质。这种作用是相当大的,并且可以解释为什么不同的去污剂会抑制这种将a ud蛋白保持在其天然状态的不同能力,因为某些可能只是不能够共溶解对于稳定性(和功能)必不可少的天然脂质。当使用透析或稀释去除洗涤剂时,与蛋白质结合的去污剂的量是特别令人关注的。此外,在大多数情况下,蛋白质一定不能暴露于过量的去污剂中,而过量的去污剂无论如何都不能令人满意地模仿“双反层”。正如 ud第4章的讨论中指出的那样,当溶液中膜蛋白的疏水性 ud区域周围存在的去污剂环/接近其溶解度极限(Psol)时,蛋白的重构就变得容易。 ud脂质也是如此:当脂质体发生时,重构很可能发生 udud过量的去垢剂也是不希望的。 ud另外,即使已知对蛋白质稳定性有不利影响的去垢剂也可以用于 u血脂增溶,因为它们的存在浓度是 uumud的。使用洗涤剂混合物 udin结晶还可以起到 udud周围 ud蛋白周围洗涤剂领的尺寸减小的作用。此外,第二种物质的存在会改变游离洗涤剂的浓度 udin洗涤剂混合物,并且在某些情况下对于形成晶体 ud是至关重要的(Koning,2003)。混合物,对于三元胶束的形成应格外小心。理想情况下,在去污剂去除之前必须完成三元混合物的平衡。 ud2.4将环糊精用于 ud高纯2D结晶 udof膜蛋白 ud第5章演示了基于 udcyclodextrin的去污剂去除二维 ud结晶的可行性。通过在不同的时间间隔简单地添加不同数量的环糊精来选择不同的动力学的可能性,这是该方法的主要优势之一。通过实施光谱学,不可能在以下条件下减慢去污剂的去除速度脂质体的发生和二维晶体的形成。 udLichtenberg等人指出。 (Lichtenberg udet等人,2000)去污剂的去除速度必须足够慢,以允许去污剂引起的囊尺寸的增长,这一过程通常非常慢。这一点很重要,因为 udone定义了洗涤剂中和的速度(与透析相反)。乍一看,人们可能会认为,就透析而言,在这方面环糊精方法没有优势。但是,通过透析去除低cmc去污剂的速度可能太慢,从而使蛋白质在其天然环境中停留的时间过长,最终促进了其沉淀。 ud在第6章中,我们介绍了一种平行的设备定量重建和2D结晶 udof膜蛋白。环糊精为实现高通量实施提供了独特的机会与当今的其他方法相比。 ud通过控制蒸发浓缩 ud并伴随去污剂的中和(以 udprevent去污剂浓缩)是有利的与市售的蛋白浓缩 ud设备相比浓缩洗涤剂胶束。此外,使用 udone蛋白制备物进行广泛筛选的可能性可确保排除由于制备 uddifferences而导致的结果不一致。通常,纯化蛋白的去污剂/去污剂和脂质浓度通常是 udill的特征,这种变异性可能是导致许多不可再现性和结晶失败的原因 ud(Wiener,2004)。 ud到目前为止,广泛筛选的使用二维晶体学中的矩阵(稀疏 udmatrix设计)受限 ud受大量实验和 ud uda严格筛选所需的蛋白质的限制。 ud所展示的机器可以部分 udcompensate弥补蛋白质中的第一个瓶颈 udstructure阐明,这是膜蛋白的过表达 udof。 2.1总结了基于第6章及以上内容中讨论的标准的筛选策略。 ud筛选效率由问题的细分 ud分成多个子问题并由其顺序筛选提供。 ud采用高通量方法,但是 udnew瓶颈的出现是因为将进行大量大量的结晶试验,因此必须对其结果进行筛选。因此,与X射线社区类似,自动 udsample制备和自动电子 ud显微镜分析的发展将为2D结晶仪提供坚实的支持。 ud结合关键值的分步识别结晶所需的不必要(和/或有效重构)与高通量筛选 udmatrices一起为膜蛋白的结构和功能不确定性的研究开辟了新的前景。现在有可能应用半理性筛选策略,这可能有助于将2D结晶从艺术转变为科学(Jap等,1992)。

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    Kaufmann Thomas Claudio;

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  • 年度 2006
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