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 non-denaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.Membrane proteins are responsible for a wide range of essential physiological processes and are involved in various diseases. Most biophysical methods for studying membrane proteins in vitro require a membrane mimicking environment to stabilize the protein. 1 The most commonly used membrane mimics are detergents, but since they often have deteriorating effects on the structure and activity of a protein, the search for a suitable detergent is an empirical and thus time-consuming process. A better mimic are phospholipid bilayers, since their biophysical properties resemble more closely those of the native membrane and they are thus more likely to maintain membrane proteins in a stable and active state. Phospholipid bilayers in the form of bicelles have been used to study membrane proteins by high resolution biophysical methods such as NMR spectroscopy 2 and x-ray crystallography. 3 However, the morphology of bicelles is complex, depending on various sample conditions, such as gerhard_wagner@hms.harvard.edu. Supporting Information Available: Experimental procedures and data on the purification, refolding and reconstitution of VDAC-1 into nanodiscs. This material is available free of charge via the Internet at http://pubs.acs.org. A promising new approach for studies of membrane proteins in a phospholipid bilayer uses high-density lipoprotein nanodiscs consisting of a central disk-shaped core of lipids, confined by two copies of an α-helical amphipathic protein. [5][6][7] These nanoscale lipid bilayers with reported diameters ranging from 9.5 to 12 nm prov...

Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation

“…Application of solution NMR methods to study the structure of membrane proteins reconstituted into phosphoplipid vesicles is hindered by the very large size of the vesicles, which effectively ranges from tens of megadaltons to gigadaltons depending on their radii and leads to broadening beyond detection of resonances from structured parts of the proteins. This problem can be alleviated by incorporation of membrane proteins into nanodiscs (30)(31)(32), which leads to effective molecular weights in the 150-400 kDa range but still requires protein perdeuteration to obtain high-quality NMR data for structured regions. In the current study, we made no attempt to observe resonances from structured regions of reconstituted synaptobrevin and focused on determining which regions of its sequence remain flexible upon anchoring to a phospholipid bilayer.…”

“…Conversely, our data show that synaptobrevin behaves very similarly on nanodiscs and on phospholipid vesicles, confirming the notion that nanodiscs provide a faithful membrane model. Although our NMR experiments of synaptobrevin in nanodiscs focused on analyzing flexible regions, recent studies have shown that good-quality NMR data can also be obtained for structured regions of membrane proteins inserted into nanodiscs (30)(31)(32). Hence, the nanodisc approach provides a promising avenue for structural studies of membrane proteins in their actual bilayer environment.…”

σ-1 Receptor at the Mitochondrial-Associated Endoplasmic Reticulum Membrane Is Responsible for Mitochondrial Metabolic Regulation

“…Because the N-terminal segment of VDAC is involved in voltage gating, it may adopt different conformations depending on the factors external to the protein (Ujwal et al, 2008). Using electron microscopy and NMR studies with a nanodisc technology, it has been demonstrated that the presence of phospholipid membrane facilitates this targeting to or interaction with different proteins (Raschle et al, 2009). Because VDAC1 knockout mice are viable (Anflous et al, 2001), it is likely that additional mechanisms promote cholesterol transport, possibly through the interaction of the -1 receptor associated with VDAC2 at the MAM region.…”

Artificial membranes for membrane protein purification, functionality and structure studies

“…A number of membrane proteins, including cytochrome P450 monooxygenase [35,44], bacteriorhodopsin [45], rhodopsin [46,47], β2-adrenergic receptor [48,49], and the human mitochondrial voltage-dependent anion channel (VDAC1) have been studied using nanodisc technology [50].…”