Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Summary

Fabrication procedures for highly magnetically responsive lanthanide ion chelating polymolecular assemblies are presented. The magnetic response is dictated by the assembly size, which is tailored by extrusion through nanopore membranes. The assemblies' magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering.

Abstract

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln³⁺) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln³⁺ (molar ratio 4:1:1) bicelles. Introduction of cholesterol (Chol-OH) and steroid derivatives in the bilayer results in another set of assemblies offering unique physico-chemical properties. For a given lipid composition, the magnetic alignability is proportional to the bicelle size. The complexation of Ln³⁺ results in unprecedented magnetic responses in terms of both magnitude and alignment direction. The thermo-reversible collapse of the disk-like structures into vesicles upon heating allows tailoring of the assemblies' dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 °C, resulting in assembly dimensions defined by the vesicle precursors. Herein, this fabrication procedure is explained and the magnetic alignability of the assemblies is quantified by birefringence measurements under a 5.5 T magnetic field. The birefringence signal, originating from the phospholipid bilayer, further enables monitoring of polymolecular changes occurring in the bilayer. This simple technique is complementary to NMR experiments that are commonly employed to characterize bicelles.

Introduction

Bicelles are disk-like polymolecular assemblies obtained from numerous lipid mixtures.^1,2,3,4,5 They are widely used for the structural characterization of membrane biomolecules by NMR spectroscopy.^6,7 However, recent efforts aim to expand the field of possible applications.^5,8,9 The most studied bicelle system is composed of a mixture of 1,2-dimyristoyl-

Protocol

1. Fabrication procedure for DMPC/DMPE-DTPA/Tm³⁺ (molar ratio 4:1:1) and DMPC/Chol-OH/DMPE-DTPA/Tm³⁺ (molar ratio 16:4:5:5) polymolecular assemblies

1. Preliminary preparations
   1. Wash all the glassware by flushing once with ethanol stabilized chloroform (>99% chloroform) and dry with compressed air.
   2. Produce 2 distinct 10 mg/mL stock solutions of DMPC and DMPE-DTPA in ethanol-stabilized chloroform (>99% chloroform), a 10 mM stock solution of Chol-OH in ethanol-stabilized chloroform (>99% chloroform) and a 10 mM stock solution of TmCl₃ in methanol.
   3. Prepare a 50 mM phosphate buffer at a pH value of....

Results

The birefringence signal of a non-extruded DMPC/DMPE-DTPA/Tm³⁺ (molar ratio 4:1:1) sample was monitored under a 5.5 T magnetic field during a heating and cooling cycle from 5 to 40 °C and back at a rate of 1 °C/min (Figure 6). The birefringence results confirmed high magnetic alignments at 5 °C with a value of 1.5 x 10^-5, twice as strong as for the reported extruded systems.^6,7

Discussion

A detailed account of how birefringence measurements were used in combination with SANS experiments to evaluate methods for generating highly magnetically responsive Ln³⁺ chelating phospholipids assemblies is in Isabettini et al.²³ The proposed fabrication protocols are also applicable for assemblies composed of the longer DPPC and DPPE-DTPA phospholipids or for those containing chemically engineered steroid derivatives in their bilayer.^11,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)	Avanti Polar Lipids	850345P	>99%
1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)	Avanti Polar Lipids	790535P	>99%
Thulium(III) chloride	Sigma-Aldrich	439649	anhydrous, powder, 99.9% trace metals basis
Dysprosium(III) chloride	Sigma-Aldrich	325546	anhydrous, powder, 99.9% trace metals basis
Ytterbium(III) chloride	Sigma-Aldrich	439614	anhydrous, powder, 99.9% trace metals basis
Chloroform	Sigma-Aldrich	319988	contains ethanol as stabilizer, ACS reagent, ≥99.8%
Methanol	Sigma-Aldrich	34860	≥99.9%
Cholesterol	Amresco	433	Ultra pure grade
D2O	ARMAR chemicals	1410	99.8 atom % D
Ultrapure water	Millipore		Synergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)
electronic pH meter	Metrohm	17440010
Whatmann Nuclepore 25 mm 100nm membrane filter	VWR	515-2028
Whatmann Nuclepore 25 mm 200nm membrane filter	VWR	515-2029
Whatmann Nuclepore 25 mm 400nm membrane filter	VWR	515-2030
Whatmann Nuclepore 25 mm 800nm membrane filter	VWR	515-2032
Whatmann Filter paper	VWR	230600
25 ml round bottom flask	VWR	201-1352	14/23 NS
3 ml glass snap-cup	VWR	548-0554	ND18, 18x30mm
2.5 ml glass syringe	Hamilton
Sodium dihydrogen phosphate dihydrate	Merk	1.06342	Salt used to make phosphate buffer
di-Sodium hydrogen phosphate	Merk	1.06586	Salt used to make phosphate buffer
Liquid Nitrogen	Carbagas	-
Pressurized Nitrogen gas	Carbagas	-	200 bar bottle
Lipid Extruder 10 ml	Lipex	-	Fully equipped with thermobarrel
High-pressure PVC tube	GR NETUM	-	must resist more than 4 MPa
Serto adaptors	Sertot	-
Nitrile gloves	VWR	-
2 ml glass pipettes	VWR	612-1702	230 mm long
Diode Laser	Newport	LPM635-25C
DSP Dual Phase Lock-in Amplifier	SRS	SR830
Photodiode Detector	Silonex Inc.	SLSD-71N5	5mm2, Silicon, photo-conductive
5.5 T Cryogenic Magnetic	Cryogenic/Oerlikon AG	-	12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head
Second order low pass filter	home-built	-	Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA
Photoelastic modulator	Hinds instruments	PEM-90
Glan-Thompson Calcite Polarizer	Newport	10GT04	25.4mm diameter
Quartz sample cuvette	Hellma	165-10-40	temperature controlled cell, 0.8 ml, 10mm path length
Temperature probe	Thermocontrol	-	Type K, 0.5mm diameter, Thermocoax
Non-polarizing mirrors	Newport	50326-1002	25.4mm
RS 232 cables	National Instruments	189284-02	For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers
BNC 50 Ω cable and connectors	National Instruments	763389-01
cFP-AI-110	National Instruments	777318-110	8-Channel Analog Voltage and Current Input Module for Compact FieldPoint
cFP-CB-1	National Instruments	778618-01	Integrated Connector Block for Wiring to Compact FieldPoint I/O
cFP-CB-3	National Instruments	778618-03	Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module
cFP-TC-120	National Instruments	777318-120	8-Channel Thermocouple Input Module for Compact FieldPoint
cFP-1804	National Instruments	779490-01	Ethernet/Serial Interface for NI Compact FieldPoint
LabView 2010	National Instruments	-
Industrial power supply	Traco Power	TCL 060-124	100-240V AC
Waterbath	Julabo	FP40-HE	refrigerated/Heating Circulator