Preparation of Lipid Nanodiscs with Lipid Mixtures.

Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Assembly of mixed lipid nanodiscs Basic Protocol 2: Assembly of nanodiscs with cholesterol Basic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements.

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

P-glycoprotein (P-gp) is a highly substrate-promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide-binding domains (NBDs) to drive transitions between inward-facing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of local dynamics has not been determined. In this work we measured the local dynamics of the IF state of P-gp in lipid nanodiscs and in detergent solution by hydrogen-deuterium (H/D) exchange MS. We observed "EX1 exchange kinetics," or bimodal kinetics, for several peptides distributed in both NBDs, particularly for P-gp in the lipid nanodiscs. Remarkably, the EX1 kinetics occurred on several time scales, ranging from seconds to hours, suggesting highly complex, and correlated, motions. The results indicate at least three distinct conformational states in the ligand-free P-gp and suggest a rough conformational landscape. Addition of excess ATP and vanadate, to favor the OF conformations, caused a generalized, but modest, decrease in H/D exchange throughout the NBDs and slowed the EX1 kinetic transitions of several peptides. The functional implications of the results are consistent with the possibility that conformational selection provides a source of substrate promiscuity.

Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs.

The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2-10-fold increases in KD in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.

Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance.

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of Biacore sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described in this unit and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody. Advantages and disadvantages of each chip type are considered.