Reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities

Abstract

The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane - monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro.

Figure 1

Schematic drawings of (A) monolayer, (B) supported lipid bilayer, (C) liposomes and (D) nanodisc. Phospholipids contain two fatty acid tails, shown in red and a hydrophilic head group, shown in blue. Light blue (A & B) and black in B represent water and a substrate respectively. Nanodiscs contain membrane scaffold proteins, shown in green.

Figure 2

Π–t adsorption isotherms of myristoylated and nonmyristoylated recoverin adsorb onto a dimyristoyl phosphatidylcholine monolayer [21].

Figure 3

(A) The reconstitution of integral membrane proteins into supported lipid bilayer. (B) Polymer-supported lipid bilayers are designed to space the lipid bilayer from the substrate [30]. Purple represents the polymer cushions which are assembled on a substrate, shown in blue.

Figure 4

Capture of His-tagged nanodiscs to Ni-NTA sensor chips followed by binding of 20 nM cholera toxin subunit B (CTB) to nanodiscs that contain (A) 0% GM1 and (B) 2% GM1. The response measured in resonance units (RU) is linearly dependent on the mass bound to the sensorchip [73].

Figure 5

Current–voltage relationship curves of (A) the intermediate K⁺-selective nuclear ionic channels in asymmetrical 150/50 mM (•) KCl and in symmetrical 150/150 mM (○) KCl conditions and (B) Cl⁻-selective nuclear ionic channels in asymmetrical 150/50 mM (○) KCl and in symmetrical 50/50 mM (□) or 150/150 mM (•) KCl conditions [74].

Figure 6

Comparsions of the ATPase activity of P-glycoprotein in nanodiscs (square) and proteoliposomes (circle). Open symbols: basal activity in the absence of drug; filled-in symbols: activity in the presence of nicardipine [76].

Figure 7

The ATPase activity of MalFGK2 was measured in nanodisc, detergent solubilized conditions and proteoliposomes in the presence of MalE or maltose [78].