Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction

Abstract

Source: Matos, A. L. L., et al. Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator. J. Vis. Exp. (2018).

This video demonstrates a dissipative microgravimetry technique to investigate the binding of proteins to lipid bilayers. Small unilamellar vesicles are added to coat the surface of the quartz sensor of a microbalance. Utilizing the calcium ion-dependent binding of the target phospholipid-binding protein to the bilayer, changes in the oscillation frequency of the sensor and the dissipation of the oscillation are monitored to determine the nature of the protein-lipid bilayer binding.

Protocol

NOTE: Buffers should be filtered using a 0.22-µm filter and degassed by a vacuum for 1 h.

1. Lipid Vesicle Preparation

1. Use 2-mL glass tubes. Dissolve each lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesterol (Chol), in a mixture of chloroform/methanol (50:50 v/v) to prepare a clear 5 mM lipid solution. Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) (PI(4,5)P₂) in a mixture of chloroform/methanol/water (20:9:1 v/v).....

Results

Table 1: Lipid composition and formation data of the SLB

Composition	ΔΔF/Hz after formation of SLBs	ΔΔD*10-6 after formation of SLB
POPC/POPS(80 : 20)	26.3 ± 0.2	0.26 ± 0.03
POPC/PI(4,5)P2(95 : 5)	26.5 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Calcium chloride	Merck	017-013-00-2	99%
Chloroform	Roth	4432.1	99%
DOPC	Avanti	850375P
EGTA	PanReac AppliChem	A0878	99%
HEPES	PanReac AppliChem	A1069
Methanol	PanReac AppliChem	A3493
PiP2	Avanti	850155P
POPC	Avanti	850457P
POPS	Avanti	840034P
Sodium chloride	PanReac AppliChem	A1149
SDS	Roth	183
Trisodium citrate	PanReac AppliChem	A3901
Equipment
Extruder Liposofast	Avestin
Qsense E4 Analyzer	Qsense
QSense Dfind	Qsense
Pump IPC 4	Ismatec	ISM 930
QSX 303 SiO2 Silicon dioxide 50nm	Qsense	QSX 303
PC Membranes 0.05μm	Avanti polar lipids	610003
OriginPro	OriginLab Corporation		Version 8 and 9