This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.
Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.
在过去的十年,五聚配体门控离子通道的结构理解(pLGIC)增长的跨越式发展,由于家庭的几名成员的高分辨率结构的众人。导致在该领域的当前进展的关键因素包括,原核pLGIC渠道发现,真核生物膜蛋白表达1-3主要进展,4-6和巨大的突破,结构的测定方法。7这些结构上提供了一个明确的共识pLGIC的三维结构的整体保护。然而,这似乎落后背后两大领域是这些渠道制剂的功能特性和通道功能的机械描述。
选通的构象变化是复杂的,发生在沿着通道的长度的60埃的距离和这些转换由广泛调制膜脂。特别是,负脂质,胆固醇和磷脂已经显示调节pLGIC 8-11的功能。尽管通道功能,这些脂质成分的确切作用尚不清楚,门完整的分子机制,需要在他们自然环境研究这些通道。定点自旋标记(SDSL)和电子顺磁共振(EPR)谱是首选在复原系统研究蛋白质动力学的技术。 EPR谱不被分子大小的限制(这是NMR)或样品的光学特性(如为荧光光谱法),由此允许在天然脂质条件重构全长构建体的测量。该技术是非常敏感的,并具有相对低的样品的要求(在微微摩尔的范围)。这两个方面使该技术非常适合于大型研究膜蛋白难以超过毫克来表达数量。
利用与定点自旋标记组合EPR谱是由韦恩哈贝尔和同事开发,并已被改编为研究的范围内的蛋白质的类型。12-24 EPR数据的已被用于研究的二级结构,在该蛋白质改变构象,膜 – 插入深度,以及蛋白质 – 蛋白质/蛋白质 – 配体相互作用。
该方法涉及在由定点诱变感兴趣位置半胱氨酸取代。为确保位点特异性标记,就必须以替代天然半胱氨酸与另一个氨基酸( 例如 ,丝氨酸)来创建一个半胱氨酸少模板。到目前为止,最流行的自旋标记是一个特定硫醇MTSL:(1-氧基-2,2,5,5-四甲基Δ3吡咯啉-3-甲基)methanethiosulfonate通过二硫键桥连接到蛋白质上。由于其高特异性,相对小的尺寸(小于色氨酸稍大),和灵活接头区域的相容性,这自旋标记已被证明具有优异的反应性,即使埋入半胱氨酸。此外,为了最大限度地提高反应性,该蛋白质的标记反应是在洗涤剂溶解的形式进行。通过尺寸排阻色谱的过量的游离自旋标记的分离后,将蛋白质重组到脂质体或限定的脂质组合物的双层模仿系统。在一般情况下,半胱氨酸诱变耐受良好的蛋白质的大部分地区,和自旋探头的相对小的尺寸会导致极小扰动的二级和三级结构。以确保该变形保留野生型功能,标记和重构通道可以通过膜片钳测量来研究。
然后将标记的官能蛋白进行光谱测量,其基本上提供三种主要类型的信息:由linesh 12,14,15,20,22,23,25-27自旋探针动力学猿分析;探头顺松弛剂无障碍;和距离的分布。27 EPR距离通过两种不同的方法进行测定。第一种是基于所述连续波(CW)技术,其中,从自旋标签之间偶极相互作用产生的频谱展宽(在8 – 20埃的距离范围内)。用于确定距离28,29的第二个是一个脉冲的EPR方法,其中,距离测量可以延长至70埃。30-34在双电子电子共振(鹿),在自旋回波振幅振荡被分析以确定的距离和距离的分布。这里自旋回波是在偶极相互作用的频率调制。总之,这些参数被用来确定蛋白质的拓扑结构,二级结构元件,和蛋白质的构象变化。
EPR谱已被证明是在一个近乎完美的环境量化膜蛋白的构象变化无与伦比的结构方法。这种方法可以让我们一窥了在高分辨率结构遮蔽X射线晶体学和低温电子显微镜蛋白质动力学的分子机制。但是,要考虑这种做法可能会影响一般适用于其他系统,并要牢记沿途的潜在实验路障的技术限制,是非常重要的。我们讨论一些这些方面的下面一起排除故障推荐的策略。
其中较常见的…
The authors have nothing to disclose.
我们非常感谢Chakrapani实验室对稿件批判性阅读和评论的现任和前任成员。 这项工作是由卫生部授予全国学院(1R01GM108921)和美国心脏协会(NCRP科学家发展赠款12SDG12070069)和SC的支持。
Site-Directed Mutagenesis and Cys mutations | |||
10x PfuUltra HF reaction buffer | Agilent Technologies | 600380-52 | |
dNTPS | New England BioLabs Inc | N0447L | 10mM each dNTP |
pfu Ultra DNA polymerase | Agilent Technologies | 600380-51 | 2.5 U/ul |
DPNI | New England BioLabs Inc | R0176S | 20,000 U/ml |
XL10 GOLD | Agilent Technologies | 200314 | |
SOC media | New England BioLabs Inc | B9020S | |
Kanamycin | Fisher Scientfic | BP905 | |
LB media | Invitrogen | 127957084 | |
Miniprep kit | QIAGEN | 27106 | |
C43 competent cells | Lucigen | 60446 | |
Expression and Purification | |||
Glucose | Fisher Scientfic | D16 | |
Tryptone | Fisher Bioreagents | BP1421-500 | |
Yeast extract | Amresco | J850 | |
Glycerol | Fisher Bioreagents | BP229 | |
K2HPO4 | Amresco | 0705 | |
KH2PO4 | Amresco | 0781 | |
IPTG (isopropyl-thio-β-galactoside) | Gold Biotechnology | I2481C25 | |
Trizma Base | Sigma Life Science | T1503 | |
NaCl | Sigma-Aldrich | S7653 | |
DNase I | Sigma Life Science | DN25 | |
PMSF | Amresco | M145 | |
Leupeptine | Amresco | J580 | |
Pepstatin | Amresco | J583 | |
DDM (n-Docecyl-β-D-Maltopyranoside) | Anatrace | D310S | |
Amylose resin | New England BioLabs Inc | E8021L | |
TCEP | Amresco | K831 | |
EDTA | Fisher Scientfic | BP118 | |
Maltose | Acros Organics | 329915000 | |
Superdex 200GL | GE Healthcare | 17-5175-01 | |
Empty polypropylene Chromatography column | BioRad | 731-1550 | |
Site-Directed Spin Labeling | |||
MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate | Toronto Reaserch chemicals Inc | O873900 | |
(1-acetoxy-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate | Toronto Reaserch chemicals Inc | A167900 | |
DMSO | J.T. Baker | 9224-01 | |
Reconstitution | |||
Asolectin lipid | Avanti polar lipids Inc | 541602C | |
Biobeads (Polystyrine beads) | Bio Rad | 152-3920 | |
Methanol | Fisher chemicals | A413 | |
FRET | |||
Fluorescein-maleimide | ThermoFisher Scientific | F-150 | |
Tetramethylrhodamine-maleimide | ThermoFisher Scientific | T-6027 | |
POPC | Avanti polar lipids Inc | 850457C | |
POPG | Avanti polar lipids Inc | 840457C | |
E.Coli polar lipid extract | Avanti polar lipids Inc | 100600C | |
HEPES | Sigma Life Science | H3375 | |
EPR measurement | |||
TPX plastic capillaries | Bruker | ER221 | |
EDDA (Ethylenediamine-N, N'-diacetic acid) | Aldrich | 158186 | |
Ni(OH)2 | Aldrich | 283622 |