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Bio-ingénierie

PIP-on-a-chip：蛋白质 - 磷酸肌醇相互作用的无标记研究

Published: July 27, 2017
doi:
10.3791/55869
, , ,
1Department of Biochemistry and Molecular Biology,The Pennsylvania State University, 2Department of Chemistry,The Pennsylvania State University

Summary

我们在微流体平台的背景下提供支持的脂质双层，以使用基于pH调节的无标记方法研究蛋白质 – 磷酸肌醇相互作用。

Abstract

许多细胞蛋白质与膜表面相互作用以影响基本的细胞过程。这些相互作用可以针对膜内的特定脂质组分，如在磷酸肌醇（PIP）的情况下），以确保特定的亚细胞定位和/或活化。已广泛研究PIP和细胞PIP结合结构域以更好地了解其在细胞生理学中的作用。我们对支持的脂质双层（SLB）进行了pH调节测定，作为研究蛋白质-PIP相互作用的工具。在这些研究中，pH敏感的邻 –磺酰罗丹明B缀合的磷脂酰乙醇胺用于检测蛋白质-PIP相互作用。当蛋白质与含PIP的膜表面结合时，调节界面电位（ 即局部pH值的变化），使探针的质子化状态发生变化。通过使用磷脂酶C delta1 Pleckstr提出了pH调节测定的成功应用的案例研究在同源性（PLC-δ1PH）结构域和磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）相互作用为例。这种相互作用的表观解离常数（ K d，app ）为0.39±0.05μM，类似于其他人获得的K d值。如前所述，PLC-δ1PH结构域是PI（4,5）P 2特异性，显示与磷脂酰肌醇4-磷酸的结合较弱，并且不与纯磷脂酰胆碱SLB结合。 PIP片上测定优于传统的PIP结合测定，包括但不限于低样品体积和无配体/受体标记要求，测试高和低亲和力膜相互作用的能力与小和大分子，提高信噪比。因此，PIP-on-a-chip方法的使用将有助于阐明各种膜相互作用的机制。此外，这种方法可能是你的确定调节蛋白质与膜相互作用的能力的治疗剂。

Introduction

无数的相互作用和生物化学过程发生在二维流体的膜表面上。真核细胞中的膜封闭的细胞器不仅在生物化学过程及其相关的蛋白质组学中而且在其脂质组成中是独特的。一类特殊的磷脂是磷酸肌醇（PIP）。即使它们包括只有1％的细胞脂质组的，它们在信号转导，自噬和膜运输至关重要的作用，其中包括1，2，3，4。通过细胞PIP激酶肌醇头部基团的磷酸化动态产生七个PIP头基是单- ，双- ，或三-磷酸化5。另外，PIP定义膜的亚细胞同一性，并且用作含有一种或多种磷酸苷酶的蛋白质/酶的专门的膜对接位点itide结合结构域，例如，pleckstrin同源（PH），PHOX同源性（PX），和底物蛋白N端同源性（ENTH）6,7。最好研究的PIP​​结合结构域之一是在高纳摩尔 – 低微摩尔范围亲和力8内与磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）特异性相互作用的磷脂酶C（PLC）-δ1PH结构域，9，10，11。

已经开发了多种定性和定量的体外方法，用于研究这些相互作用的机制，热力学和特异性。在最常用的PIP结合测定中，表面等离子体共振（SPR），等温量热法（ITC），核磁共振（NMR）光谱，脂质体浮选/沉降测定和脂质印迹（脂肪印迹/ PIP条）12，13。即使这些被广泛使用，它们都具有许多缺点。例如，SPR，ITC，和NMR需要大量的样品，昂贵的仪器，和/或受过训练的人员12，13的。一些测定法形式，例如基于抗体的脂质的印迹利用的PIP的水可溶形式，并在非生理方式12，14，15，16呈现它们。此外，脂质印迹不能可靠定量，它们经常导致假阳性/阴性的观察12，17，18。为了克服这些挑战并改进当前的工具集，在上下文中基于支持的脂质双层（SLB）建立了一种新的无标记方法 icrofluidic平台，将其成功地应用于蛋白质-PIP相互作用的研究（ 图1）19。

用于检测蛋白质-PIP相互作用的策略是基于pH调节感测。这涉及一种pH敏感的染料，其具有直接与磷脂酰乙醇胺脂质头部组20缀合的邻 –磺酰罗丹明B（ o SRB）。 o SRB-POPE探针（ 图2A ）在低pH下是高度荧光的，在高pH下淬灭，在7.5mol％含PI（4,5）P 2的SLB中具有约6.7的pKa（ 图5B ）。 PLC-δ1PH结构域已被广泛使用，用于验证蛋白质-PIP结合方法由于其对PI（4,5）P 2（ 图5A）高特异性21,22“> 23，24，25 .Hence，我们的理由是，PLC-δ1PH结构域可以用来测试其结合至PI（4,5）P 2通过PIP-上的单芯片测定法。该PH结构域构建体在此研究中使用具有净正电荷（等电点8.4），并且因此吸引OH –离子（ 图5C）在结合PI（4,5）P 2含SLBs中，PH结构域所带来的OH –离子的膜表面，这反过来又调制所述界面电势并转移öSRB-POPE（ 图5C）26，随着PH结构域浓度的函数，荧光被猝灭（ 图6A）的质子化状态。最后，将归一化的数据是适合于结合等温线以确定PH结构域-P1（4,5）P 2相互作用的亲和力（ 图6B ， 6C ）。/ P>

在这项研究中，提供了详细的方案，以在微流体平台内进行蛋白质结合含PIP的SLBs。该协议使读者将微流体装置和囊泡制备装配成SLB形成和蛋白质结合。此外，提供了用于提取PLC-δ1PH域-PI（4,5）P 2相互作用的亲和度信息的数据分析方向。

Protocol

1.清洁玻璃盖板稀释7倍的清洁溶液（参见材料表 ）用100毫米深硼硅酸盐玻璃皿的去离子水将其稀释7倍，并将其在平板热板上加热至95℃，持续20分钟，或直到混浊溶液变澄清。 注意：解决方案将会很热，请注意避免身体受伤。 7x清洁溶液是[氧化 – （氧化（膦酰氧基）磷酰基]氧磷酰基]磷酸六钠，2-（2-丁氧基乙氧基）乙醇，1,4-双（2-乙基己氧基）-1,4-二氧代丁烷钠-2-磺酸?…

Representative Results

我们使用pH调节测定法研究了PIP-on-a-chip微器件中的PLC-δ1PH域-PI（4,5）P 2相互作用（ 图1 ）。通过详细的方案，我们演示了如何准备和组装微流体装置组件，制备小单层囊泡（SUV）（ 图2 ），在装置内形成SLB（ 图3 ），并测试蛋白质与含有PIP的SLB的结合。 图4中描绘了典型的SLB…

Discussion

每个PIP变体虽然浓度低，但存在于特定细胞器的细胞溶质表面，其中它们有助于建立独特的物理组成和细胞器膜1的功能特异性。一个的PIP的最重要的用途是作为一个特定的对接平台需要特定亚细胞定位和/或活化6,7蛋白质的众人。由于其在细胞生理学和疾病中的作用，在生理学相关的背景下研究体外蛋白质-PIP相互作用的能力是?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

部分支持DS和CEC的授权AI053531（NIAID，NIH）; SS和PSC由授权N00014-14-1-0792（ONR）支持。

Materials

Coverslip
Glass Coverslips: Rectangles Fisher Scientific 12-544B 22 x 40 x 0.16 – 0.19 mm, No. 1 1/2; Borosilicate Glass
7X Cleaning Solution MP Biomedicals 976670 Detergent
PYREX Crystallizing Dish Corning 3140-190 Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)
Sentry Xpress 2.0 Paragon Industries SC-2 Kiln
Name Company Catalog Number Comments
PDMS
Sylgard 184 Silicone Elastomer Kit Dow Corning  4019862 Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives
PYREX Desiccator VWR 89134-402 Vacuum Rated
Biopsy punch Harris 15110-10 Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative
Name Company Catalog Number Comments
Device
Plasma Cleaning System PlasmaEtch PE25-JW 2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)
Digital Hot Plate Benchmark H3760-H Purchased through Denville Scientific (Cat. No. 1005640)
Frosted Micro Slides VWR 48312-003 Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144
Name Company Catalog Number Comments
Mold
AutoCAD Autodesk v.2016 Drafting software for the photomask design
Photomask CAD/Art Services N/A Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services
Silicone Wafers University Wafer 1575 Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness
SU-8 50 MicroChem Corp. N/A Negative Tone Photoresist; Penn State Nanofabrication Facility Property
SU-8 Developer MicroChem Corp. N/A Penn State Nanofabrication Facility Property
Name Company Catalog Number Comments
SUV
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Avanti Polar Lipids 850457C POPC
L-α-phosphatidylinositol-4-phosphate Avanti Polar Lipids 840045X PI4P
L-α-phosphatidylinositol-4,5-bisphosphate  Avanti Polar Lipids 840046X PI(4,5)P2
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Avanti Polar Lipids 850757C POPE; Required for the synthesis of oSRB-POPE
Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers) ThermoFisher Scientific L-20 Required for the synthesis of oSRB-POPE
pH Sensitive Fluorescent Lipid Probe (oSRB-POPE) In-house N/A In-house Synthesis (Huang D. et al. 2013)
Glass Scintillation Vial VWR 66022-065 20 mL volume capacity
Aquasonic 250D VWR N/A Ultrasonic Water Bath
Nuclepore Track-Etched Membranes Whatman 110605 Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich
Chloroform VWR CX1054-6 HPLC grade
LIPEX Extruder Transferra Nanosciences T.001 LIPEX 10 mL Thermobarrel Extruder
Viscotek 802 DLS Malvern Instruments N/A Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property
Name Company Catalog Number Comments
Data Analysis
GraphPad Prism GraphPad Software v.6 Curve-fitting software for data analysis
Name Company Catalog Number Comments
Microscope
Axiovert 200M Epifluorescence Microscope Carl Zeiss Microscopy N/A Microscope
AxioCam MRm Camera Carl Zeiss Microscopy N/A Camera
X-Cite 120 Excelitas Technologies N/A Light Source
Alexa 568 Filter Set Carl Zeiss Microscopy N/A Ex/Em 576/603 nm
AxioVision LE64 v.4.9.1.0 Software Carl Zeiss Microscopy N/A Image Processing Software
Name Company Catalog Number Comments
Other
Tips VWR 10034-132 200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel
Tips VWR 53509-070 10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel
Orion Star A321 pH meter Thermo Scientific STARA3210 pH meter
Orion micro pH probe Thermo Scientific 8220BNWP micro pH probe
N-(2-Hydroxyethyl)-Piperazine-N'-(2-Ethanesulfonic Acid) VWR VWRB30487 HEPES, Free Acid
Sodium Chloride VWR BDH8014-2.5KGR NaCl
Tubing Allied Wire & Cable TFT-200-24 N Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color
Nitrogen Gas – Industrial Praxair N/A Local Provider
Oxygen Gas – Industrial Praxair N/A Local Provider
Liquid Nitrogen Praxair N/A Local Provider
DOWNLOAD MATERIALS LIST

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Tags

Protein-phosphoinositide InteractionsLabel-freeMembrane InteractionsPIP-on-a-chip AssayPDMSPhosphatidylcholinePhosphatidylinositol 45-bisphosphatePH-sensitive Fluorescent ProbeLipid VesiclesLabel-free MonitoringBiologically RelevantTherapeutic TargetsMembrane Proteins

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Shengjuler, D., Sun, S., Cremer, P. S., Cameron, C. E. PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions. J. Vis. Exp. (125), e55869, doi:10.3791/55869 (2017).
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