Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs

Abstract

Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under nondenaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.

Figure 1

Electron microscopy of VDAC-1 in DMPC nanodiscs. (A) Representative image of negatively stained nanodiscs containing VDAC channels. The stain-filled channels appear as dark spots in the brighter nanodiscs. The scale bar is 50 nm. (B) Representative class averages showing the variation of the nanodisc population in size (averages 1 to 4) and in the number of VDAC channels per nanodisc (averages 5 to 16). The side length of the individual panels is 28.8 nm.

Figure 2

2D [¹⁵N,¹H]-TROSY spectrum of [U-²H,¹⁵N]-VDAC-1 in DMPC nanodiscs (blue) and in LDAO micelles (red).

Figure 3

NADH binding of VDAC-1. Selected regions of 2D [¹⁵N,¹H]-TROSY spectra of VDAC-1 in (A,B) nanodiscs and (C,D) LDAO micelles. The blue and red spectra correspond to Figure 2, recorded in the absence of NADH. The orange spectra were recorded after addition of NADH. Sequence-specific resonance assignments of VDAC-1 in LDAO micelles are indicated. Residues G244 and V237, which are part of the NADH binding site, are printed bold.