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Materials

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Biochemistry

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Published: November 30th, 2022

DOI:

10.3791/64340

1School of Chemistry, Chemical Engineering, and Biotechnology, Nanyang Technological University, 2State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, 3Institute for Digital Molecular Analytics and Science, Nanyang Technological University
* These authors contributed equally

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

Membrane curvature is a critical physical parameter of a cell that occurs in a variety of cellular processes such as morphogenesis, cell division, and cell migration1. It is widely recognized now that membrane curvature is beyond a simple result of cellular events; instead, it has emerged as an effective regulator of protein interactions and intracellular signaling. For example, several proteins involved in clathrin-mediated endocytosis were found to preferentially bind to the curved membrane, resulting in the formation of a hotspot for endocytosis2. There are many different causes of membrane deformation such as membran....

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1. Cleaning of nanochip

  1. Place the nanochip in a 10 mL beaker with the patterned side facing up.
    NOTE: This quartz nanochip has been fabricated via electron beam lithography as described before21. The geometry and arrangement of the nanostructure on the chip can be custom designed. The sizes of the gradient nanobars used here are 2000 nm in length, 600 nm in height, and 100 to 1000 nm in width (100 nm step-set).
  2. Carefully add 1 mL of 98% sulfur.......

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Nanobar design is recommended for probing positive curvature sensing proteins, which contains a half circle at each end with curvature defined by the nanobar width and one flat/zero curvature control locally at the center (Figure 2A,B). Successful formation of the SLB on nanobars results in evenly distributed lipid marker signals across the entire nanobar surface as shown in Figure 2C. Signals from multiple nanobars can be combined by averaging .......

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The nanobar-SLB system described here offers a unique combination of the advantages in several existing in vitro assays. It efficiently reveals the preferential binding of proteins to highly curved membranes as the liposome floatation or sedimentation assay but requires much fewer samples and offers more accurately defined curvature on individual nanobars8,29. It also offers a wide range of precisely controlled curvature for simultaneous comparison as th.......

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We thank Nanyang NanoFabrication Centre (N2FC) and the Centre for Disruptive Photonic Technologies (CDPT) at Nanyang Technological University (NTU) for supporting nanostructure fabrication and SEM imaging, the Protein Production Platform (PPP) at the School of Biological Sciences NTU for protein purification, and the School of Chemical and Biomedical Engineering NTU for the confocal microscope. This work is funded by the Singapore Ministry of Education (MOE) (W. Zhao, RG112/20, RG95/21, and MOE-T2EP30220-0009), the Institute for Digital Molecular Analytics and Science (IDMxS) supported by MOE funding under the Research Centres of Excellence scheme (W. Zhao), the Human....

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NameCompanyCatalog NumberComments
Anhydrous EthanolSigma-Aldrich100983
Aluminum foilDiamondRN0879999FU
Amber VialSigma-Aldrich27115-U
Brain PS: L-α-phosphatidylserine (Brain, Porcine) (sodium salt)Avanti Polar Lipids, Inc.840032
10 mL BeakerSchott-DuranSCOT211060804
50 mL BeakerSchott-DuranSCOT211061706
1000 mL BeakerSchott-DuranSCOT211065408The second container 
ChloroformSigma-AldrichV800117
Cotton budsWatsons
18:1 DGS-NTA(Ni): 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)Avanti Polar Lipids, Inc.790404
Egg PC: L-α-phosphatidylcholine (Egg, Chicken)Avanti Polar Lipids, Inc.840051
F-BARProtein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, SingaporeProteins and peptide
F-BAR+IDRProtein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, SingaporeProteins and peptide
GFPProtein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, SingaporeProteins and peptide
GFP-HisProtein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, SingaporeProteins and peptide
GraphPad PrismGraphPadV9.0.0
Hydrogen Peroxide, 30% (Certified ACS)Thermo ScientificH325-500
IDR from human FBP17Sangon Biotech (Shanghai) Co., Ltd.
ImageJNational Institutes of Health1.50d
Laser Scanning Confocal MicroscopyZeiss LSM 800 with Airyscan100x (N.A.1.4) oil objective.
MethanolFisher scientific10010240
Mini-extuder Avanti Polar Lipids, Inc.610000-1EA
1.5 mL MicrotubesGreiner616201
MATLABMathworksR2018b
Nuclepore Hydrophilic Membrane,0.1 μmWhatman800309
Phosphate Bufferen Saline (PBS)Life Technologies Holdings Pte Ltd.70013
Polydimethylsiloxane (PDMS) BaseDow Corning CorporationSYLGARD 184
Polydimethylsiloxane (PDMS) CrosslinkerDow Corning CorporationSYLGARD 184
Plasma CleanerHARRICK PLASMAPDC-002-HP
Quartz NanochipDonghai County Alfa Quartz Products CO., LTD
Sodium Hydroxide Sigma-Aldrich795429
Sulfuric acidSigma-Aldrich258105
Texas Red DHPE: Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium SaltLife Technologies Holdings Pte Ltd.T1395MP
TweezerGooiPDC-002-HP
Ultrasonic CleanersElmaD-78224
VoterxScientific IndustriesG560E
Vacuum DesiccatorNUCERITE5312
Water BathJulaboTW8

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