Human Voltage-dependent Anion Channel Research Articles

Defining the roles and regulation of the mitochondrial VDAC isoforms one molecule at a time.

The voltage-dependent anion channel (VDAC) is recognized as the key metabolite conduit in the mitochondrial outer membrane and regulator for fluxes of water-soluble metabolites, ions, and polypeptides. The uniqueness of VDAC arises from its position at the interface between the mitochondria and the cytosol making VDAC an important cellular communication hub. VDAC has three isoforms in mammals: VDAC1, 2, and 3 which share high sequence similarity, indicative of a similar pore-forming structure. All three VDAC isoforms form similar highly conductive, anion-selective, voltage-gated channels when reconstituted into planar lipid membranes implying that they can concurrently facilitate the transport of the negatively charged respiratory metabolites. However, cellular model systems and isoform-specific knock-out mice demonstrate distinctive functional roles for each VDAC isoform. We explored the biophysical basis for these differences by reconstituting human, mouse, and zebrafish VDAC isoforms in planar lipid bilayers and performing single channel electrophysiology. We have uncovered that interactions with cytosolic regulators, such as the Parkinson's disease associated alpha-synuclein, vary in their on-rates by two-orders of magnitude between each isoform. Furthermore, each isoform varies in their ability translocate synuclein across the lipid bilayer. Exploring the calcium transport properties of each isoform, we show that VDAC3 has the highest calcium permeability. We demonstrate that VDAC2 can dynamically switch between channel properties of VDAC1 and VDAC3, and explored the structural basis for its unique plasticity using mutational and structural analysis and molecular dynamics simulations. We find that both VDAC2's N-terminal extension and cysteine residues contribute to, but are not sufficient to explain VDAC2's striking behavior. Our results suggest that tuning of beta-barrel and loop flexibility between the isoforms could define their biophysical properties and help to explain distinct physiological roles.

The Molecular Mechanism of Human Voltage-Dependent Anion Channel 1 Blockade by the Metallofullerenol Gd@C82(OH)22: An In Silico Study.

The endohedral metallofullerenol Gd@C82(OH)22 has been identified as a possible antineoplastic agent that can inhibit both the growth and metastasis of cancer cells. Despite these potentially important effects, our understanding of the interactions between Gd@C82(OH)22 and biomacromolecules remains incomplete. Here, we study the interaction between Gd@C82(OH)22 and the human voltage-dependent anion channel 1 (hVDAC1), the most abundant porin embedded in the mitochondrial outer membrane (MOM), and a potential druggable target for novel anticancer therapeutics. Using in silico approaches, we observe that Gd@C82(OH)22 molecules can permeate and form stable interactions with the pore of hVDAC1. Further, this penetration can occur from either side of the MOM to elicit blockage of the pore. The binding between Gd@C82(OH)22 and hVDAC1 is largely driven by long-range electrostatic interactions. Analysis of the binding free energies indicates that it is thermodynamically more favorable for Gd@C82(OH)22 to bind to the hVDAC1 pore when it enters the channel from inside the membrane rather than from the cytoplasmic side of the protein. Multiple factors contribute to the preferential penetration, including the surface electrostatic landscape of hVDAC1 and the unique physicochemical properties of Gd@C82(OH)22. Our findings provide insights into the potential molecular interactions of macromolecular biological systems with the Gd@C82(OH)22 nanodrug.

Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel.

Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.

Cellular Interactome of Mitochondrial Voltage-Dependent Anion Channels: Oligomerization and Channel (Mis)Regulation

Voltage-dependent anion channels (VDACs) of the outer mitochondrial membrane are known conventionally as metabolite flux proteins. However, research findings in the past decade have revealed the multifaceted regulatory roles of VDACs, from governing cellular physiology and mitochondria-mediated apoptosis to directly regulating debilitating cancers and neurodegenerative diseases. VDACs achieve these diverse functions by establishing isoform-dependent stereospecific interactomes in the cell with the cytosolic constituents and endoplasmic reticulum complexes, and the machinery of the mitochondrial compartments. VDACs are now increasingly recognized as regulatory hubs of the cell. Not surprisingly, even the transient misregulation of VDACs results directly in mitochondrial dysfunction. Additionally, human VDACs are now implicated in interaction with aggregation-prone cytosolic proteins, including Aβ, tau, and α-synuclein, contributing directly to the onset of Alzheimer's and Parkinson's diseases. Deducing the interaction dynamics and mechanisms can lead to VDAC-targeted peptide-based therapeutics that can alleviate neurodegenerative states. This review succinctly presents the latest findings of the VDAC interactome, and the mode(s) of VDAC-dependent regulation of biochemical physiology. We also discuss the relevance of VDACs in pathophysiological states and aggregation-associated diseases and address how VDACs will facilitate the development of next-generation precision medicines.

NMR Spectroscopic Studies of Ion Channels in Lipid Bilayers: Sample Preparation Strategies Exemplified by the Voltage Dependent Anion Channel.

We describe approaches for the preparation of membrane proteins in detergent micelles and lipid bilayers for solution and magic angle spinning NMR studies, respectively, as exemplified by the human voltage dependent anion channel 1 (hVDAC1). Here, we report protocols for the preparation of homogenous samples of recombinant hVDAC1 in detergent micelles and lipid two-dimensional crystals yielding high resolution NMR spectra. Procedures are described for the recombinant production of stable-isotope labeled hVDAC1 in E. coli, the isolation of hVDAC1 from inclusion bodies and the refolding into detergent micelles, as well as the reconstitution of hVDAC1 into lipids to form 2D crystals.

Evolutionary selection of a 19-stranded mitochondrial β-barrel scaffold bears structural and functional significance

Transmembrane β-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane β-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

The Study of HIV-1 Vpr-Membrane and Vpr-hVDAC-1 Interactions by Graphene Field-Effect Transistor Biosensors.

The viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1) is involved in many cellular processes during the viral life cycle; however, its associated mechanisms remain unclear. Here, we designed an Escherichia coli expression construct to achieve a milligram yield of recombinant Vpr. In addition, we fabricated a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to study the interaction between Vpr and its interaction partners. The Dirac point of the SLB/G-FET was observed to shift in response to the binding of Vpr to the SLB. By fitting the normalized shift of the Dirac point as a function of Vpr concentration to the Langmuir adsorption isotherm equation, we could extract the dissociation constant (Kd) to quantify the Vpr binding affinity. When the 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) membrane was used as the SLB, the dissociation constant was determined to be 9.6 ± 2.1 μM. In contrast, only a slight shift of the Dirac point was observed in response to the addition of Vpr when the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane was used as the SLB. Taking advantage of the much weaker binding of Vpr to the DOPC membrane, we prepared a human voltage-dependent anion channel isoform 1 (hVDAC-1)-embedded DOPC membrane as the SLB for the G-FET and used it to determine the dissociation constant to be 5.1 ± 0.9 μM. In summary, using the clinically relevant Vpr protein as an example, we demonstrated that an SLB/G-FET biosensor is a suitable tool for studying the interaction between a membrane-associated protein and its interaction partners.

Do folding elements trade‐off with function in the human mitochondrial metabolite transporter?

In humans, transmembrane β‐barrels are found exclusively in the outer mitochondrial membrane (OMM). They comprise metabolite transporters, translocases, and chaperones, which together control cellular homeostasis. The metabolite transporters — known as voltage‐dependent anion channels (VDACs) – are the most abundant OMM barrels. They are 19‐stranded β‐barrel structures that also form important pharmacological targets due to their roles in metabolite flux, steroidogenesis, gametogenesis, and ROS regulation. Despite long‐standing efforts, molecular factors that regulate VDAC function and folding at the atomistic level are not known. Here, we map the folding nucleus and the assembly pathway of human VDAC isoform 2 in phosphocholine vesicles. We carry out a comprehensive measurement of hVDAC2 folding kinetics, and equilibrium thermodynamic contribution, of each of the 270 non‐Ala residues of the 294‐residue barrel. Interestingly, we obtain a two‐state thermodynamic equilibrium that is attained through two detectable on‐pathway early folding intermediates, which are assembled in milliseconds – seconds. These intermediates undergo slow conformational restructuring to form the folded VDAC2 barrel. We find that specific charged residues that form a part of these early intermediates also positively regulate the gating characteristics of VDAC2. This observation is unprecedented, as directed evolution of protein sequences in favor of function usually place a stability burden on its structure that additionally impedes protein assembly. Our findings provide molecular insight on the assembly pathway of human VDAC2, and directly link these folding elements with VDAC2 function as the metabolite transporter of human mitochondria.Support or Funding InformationThis work was supported by the Wellcome Trust/DBT India Alliance Fellowship [grant number IA/I/14/1/501305] awarded to RM.

A size-modified poisson-boltzmann ion channel model in a solvent of multiple ionic species: Application to voltage-dependent anion channel.

We present a new size-modified Poisson-Boltzmann ion channel (SMPBIC) model and use it to calculate the electrostatic potential, ionic concentrations, and electrostatic solvation free energy for a voltage-dependent anion channel (VDAC) on a biological membrane in a solution mixture of multiple ionic species. In particular, the new SMPBIC model adopts a membrane surface charge density and a natural Neumann boundary condition to reflect the charge effect of the membrane on the electrostatics of VDAC. To avoid the singularity difficulties caused by the atomic charges of VDAC, the new SMPBIC model is split into three submodels such that the solution of one of the submodels is obtained analytically and contains all the singularity points of the SMPBIC model. The other two submodels are then solved numerically much more efficiently than the original SMPBIC model. As an application of this SMPBIC submodel partitioning scheme, we derive a new formula for computing the electrostatic solvation free energy. Numerical results for a human VDAC isoform 1 (hVDAC1) in three different salt solutions, each with up to five different ionic species, confirm the significant effects of membrane surface charges on both the electrostatics and ionic concentrations. The results also show that the new SMPBIC model can describe well the anion selectivity property of hVDAC1, and that the new electrostatic solvation free energy formula can significantly improve the accuracy of the currently used formula. © 2019 Wiley Periodicals, Inc.

Structural characterization of the human membrane protein VDAC2 in lipid bilayers by MAS NMR.

The second isoform of the human voltage dependent anion channel (VDAC2) is a mitochondrial porin that translocates calcium and other metabolites across the outer mitochondrial membrane. VDAC2 has been implicated in cardioprotection and plays a critical role in a unique apoptotic pathway in tumor cells. Despite its medical importance, there have been few biophysical studies of VDAC2 in large part due to the difficulty of obtaining homogeneous preparations of the protein for spectroscopic characterization. Here we present high resolution magic angle spinning nuclear magnetic resonance (NMR) data obtained from homogeneous preparation of human VDAC2 in 2D crystalline lipid bilayers. The excellent resolution in the spectra permit several sequence-specific assignments of the signals for a large portion of the VDAC2 N-terminus and several other residues in two- and three-dimensional heteronuclear correlation experiments. The first 12 residues appear to be dynamic, are not visible in cross polarization experiments, and they are not sufficiently mobile on very fast timescales to be visible in 13C INEPT experiments. A comparison of the NMR spectra of VDAC2 and VDAC1 obtained from highly similar preparations demonstrates that the spectral quality, line shapes and peak dispersion exhibited by the two proteins are nearly identical. This suggests an overall similar dynamic behavior and conformational homogeneity, which is in contrast to two earlier reports that suggested an inherent conformational heterogeneity of VDAC2 in membranes. The current data suggest that the sample preparation and spectroscopic methods are likely applicable to studying other human membrane porins, including human VDAC3, which has not yet been structurally characterized.

The human Bcl-2 family member Bcl-rambo and voltage-dependent anion channels manifest a genetic interaction in Drosophila and cooperatively promote the activation of effector caspases in human cultured cells

We previously reported that the Bcl-2 family member human Bcl-rambo, also known as BCL2L13, induces apoptosis in human embryonic kidney 293T cells. Mouse Bcl-rambo has recently been reported to mediate mitochondrial fragmentation and mitophagy. In the present study, we showed that the transfection of human Bcl-rambo and its microtubule-associated protein light chain 3-interacting region motif mutant (W276A/I279A) caused mitochondrial fragmentation and the perinuclear accumulation of fragmented mitochondria in human lung adenocarcinoma A549 cells. In comprehensive screening using the Drosophila model in which human Bcl-rambo was ectopically expressed in eye imaginal discs, voltage-dependent anion channels (VDAC), also known as mitochondrial porin, were found to manifest a genetic interaction with human Bcl-rambo. In addition to human adenine nucleotide translocase (ANT) 1 and ANT2, the human Bcl-rambo protein bound to human VDAC1, albeit to a lesser extent than ANT2. Moreover, human VDAC1 and human VDAC2 in particular promoted the activation of effector caspases only when they were co-expressed with human Bcl-rambo in 293T cells. Bcl-rambo induced the perinuclear accumulation of fragmented mitochondria by the knockdown of VDAC1, VDAC2, and VDAC3 in A549 cells. Thus, the present study revealed that human Bcl-rambo and VDAC cooperatively promote the activation of effector caspases in human cultured cells.

Helix-strand interaction regulates stability and aggregation of the human mitochondrial membrane protein channel VDAC3.

Voltage-dependent anion channels (VDACs) are β-sheet-rich transmembrane β-barrels that are vital for metabolite transport across the mitochondrial membrane. Under cellular stress, human VDACs hetero-oligomerize and coaggregate with proteins that can form amyloidogenic and neurodegenerative deposits, implicating a role for VDACs in proteotoxicity. However, whether VDACs possess intrinsic interaction sites that can lead to protein aggregation is not known. Here, we couple a systematic thiol replacement strategy with far-UV circular dichroism spectropolarimetry and UV scattering spectroscopy to map aggregation-prone regions of human VDACs, using isoform 3 as our model VDAC. We show that the region comprising strands β7-β9 is highly aggregation prone. Further, we find that an α1-β7-β9 interaction (involving the hVDAC3 N-terminal α1 helix) can lower protein aggregation, whereas perturbations of this interaction promote VDAC aggregation. We also show that hVDAC3 aggregation proceeds via a partially unfolded structure. Our findings allow us to propose a plausible mechanism for the role of human VDACs in forming proteotoxic aggregates in the cell. The key target sites on VDACs-strands β7-β9-may be useful for developing VDAC aggregation inhibitors.

Potential blockade of the human voltage-dependent anion channel by MoS2 nanoflakes.

Despite significant interest in molybdenum disulfide (MoS2) nanomaterials, particularly in biomedicine, their biological effects have been understudied. Here, we explored the effect of MoS2 nanoflakes on the ubiquitous mitochondrial porin voltage-dependent anion channel (VDAC1), using a combined computational and functional approach. All-atomic molecular dynamics simulations suggest that MoS2 nanoflakes make specific contact interactions with human VDAC1. We show that the initial contacts between hVDAC1 and the nanoflake are hydrophobic but are subsequently enhanced by a complex interplay of van der Waals (vdW), hydrophobic and electrostatic interactions in the equilibrium state. Moreover, the MoS2 nanoflake can insert into the lumen of the hVDAC1 pore. Free-energy calculations computed by the potential of mean force (PMF) verify that the blocked configuration of the MoS2-hVDAC1 complex is more energetically favorable than the non-blocked binding mode. Consistent with these predictions, we showed that MoS2 depolarizes the mitochondrial membrane potential (Ψm) and causes a decrease in the viability of mammalian tissue culture cells. These findings might shed new light on the potential biological effect of MoS2 nanomaterials.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Human Mitochondrial VDAC Functionality Governs Scaffold Stability

ATP transport across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDACs). The three human VDAC isoforms share a similar primary sequence but differ in their interactome, and thereby, show antagonistic physiological functions. The anti-apoptotic and regulatory role of human VDAC2 (hV2) isoform must therefore originate from subtle variations in its primary sequence. However, the molecular details of how stability and function are regulated in hV2, and its impact on protein-protein interaction, are unknown. Here, we show that the 19-stranded hV2 barrel has evolutionarily retained an energetically suboptimal sequence for superior channel gating and voltage sensing. We find that residues in the N- and C-terminal zones, comprising strands β2-β8 and β17-β18, lower the scaffold stability by 1.0-3.0 kcal/mol. Furthermore, strands β5-β10 and β17 possess sequences that are intrinsically prone to association and aggregation. However, hV2 trades-off stability and aggregation for function. Residues in the N-terminal zone are important for voltage-dependent channel gating characteristics that are essential for metabolite transport. Our results demonstrate how energetic contribution of specific residues in hV2 links inversely to its functional importance. We conclude that the evolutionary selection of hV2 sequence for channel function and regulation is at the expense of innate barrel stability. To our knowledge, this is the first study to establish stability-function trade-off in a human β-barrel membrane protein. We propose that the antagonistic behavior of the three VDAC isoforms resides in subtle differences in the molecular elements that we have identified. These molecular elements decide thermodynamic stability, function, and the association network of the VDAC channels in the mitochondrial outer membrane.

Idiopathic male infertility in the Han population in China is affected by polymorphism in the VDAC2 gene.

BackgroundIt has been proved that human voltage-dependent anion channel 2 (VDAC2) plays a significant role in sperm function and male fertility. This study was primarily aimed at exploring whether VDAC2 is a risk factor for idiopathic male infertility.ResultsWe determined a significantly increased risk of idiopathic infertility with abnormal semen parameters in association with the variant rs2804535 and a decreased risk of idiopathic infertility with abnormal semen parameters in association with the variant rs11001334. However, among subjects with normal semen parameters, no significant differences could be found in these genotypes. Moreover, we could not find any differences in the variants rs7896741 and rs1259503, which showed no risk of male infertility, whether normal or abnormal.Materials and methodsAll of the experimental subjects, including 523 men who cannot conceive children and 277 fertile controls, underwent complete historical and physical examinations. Each participant donated an ejaculate for semen analysis and 5 ml of peripheral blood for genomic DNA extraction. A computer-assisted semen analysis system was used for the semen analysis. Four single-nucleotide polymorphisms were identified and analyzed using TaqMan SNP Genotyping Assays.ConclusionsThe result shows that the relationships between different variants in the VDAC2 gene and male fertility differ, and the individuals who carry those variants may have a decreased or increased risk of abnormal semen parameters associated with male infertility.

Molecular Plasticity of the Human Voltage-Dependent Anion Channel Embedded Into a Membrane

The voltage-dependent anion channel (VDAC) regulates the flux of metabolites and ions across the outer mitochondrial membrane. Regulation of ion flow involves conformational transitions in VDAC, but the nature of these changes has not been resolved to date. By combining single-molecule force spectroscopy with nuclear magnetic resonance spectroscopy we show that the β barrel of human VDAC embedded into a membrane is highly flexible. Its mechanical flexibility exceeds by up to one order of magnitude that determined for β strands of other membrane proteins and is largest in the N-terminal part of the β barrel. Interaction with Ca(2+), a key regulator of metabolism and apoptosis, considerably decreases the barrel's conformational variability and kinetic free energy in the membrane. The combined data suggest that physiological VDAC function depends on the molecular plasticity of its channel.

VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore

The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2.

The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.