TY - JOUR

T1 - Peptide disc mediated control of membrane protein orientation in supported lipid bilayers for surface-sensitive investigations

AU - Luchini, Alessandra

AU - Tidemand, Frederik Grønbæk

AU - Johansen, Nicolai Tidemand

AU - Campana, Mario

AU - Sotres, Javier

AU - Ploug, Michael

AU - Cárdenas, Marité

AU - Arleth, Lise

PY - 2019

Y1 - 2019

N2 - In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvi-ronment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable plat-form to investigate membrane proteins by a broad range of surface-sensitive techniques such as Neutron Reflectometry (NR), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR), Atomic Force Microscopy (AFM) and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challeng-ing. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to medi-ate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and Tissue Factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface and the peptide mol-ecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of mem-brane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile and we show its general applicability by characterizing with the above mentioned surface sensitive techniques two dif-ferent membrane proteins, which were efficiently loaded in the supported bilayers with ~0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins, and for preparation of suitable platforms for drug testing or biosen-sor development.

AB - In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvi-ronment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable plat-form to investigate membrane proteins by a broad range of surface-sensitive techniques such as Neutron Reflectometry (NR), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR), Atomic Force Microscopy (AFM) and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challeng-ing. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to medi-ate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and Tissue Factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface and the peptide mol-ecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of mem-brane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile and we show its general applicability by characterizing with the above mentioned surface sensitive techniques two dif-ferent membrane proteins, which were efficiently loaded in the supported bilayers with ~0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins, and for preparation of suitable platforms for drug testing or biosen-sor development.

U2 - 10.1021/acs.analchem.9b04125

DO - 10.1021/acs.analchem.9b04125

M3 - Journal article

C2 - 31769649

VL - 92

SP - 1081

EP - 1088

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 1

ER -