The N‑Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities Carlo Guardiani, † Mariano Andrea Scorciapino,* ,‡,§ Giuseppe Federico Amodeo, ∥ Joze Grdadolnik, ⊥ Giuseppe Pappalardo, # Vito De Pinto, @ Matteo Ceccarelli, †,§ and Mariano Casu ∥ † Department of Physics, University of Cagliari, 09042 Monserrato, Italy ‡ Department of Biomedical Sciences, Biochemistry Unit, University of Cagliari, 09042 Monserrato, Italy § Istituto Officina dei Materiali del Consiglio Nazionale delle Ricerche (IOM-CNR), UOS, Cagliari, Italy ∥ Department of Chemical and Geological Sciences, University of Cagliari, 09042 Monserrato, Italy ⊥ National Institute of Chemistry, Ljubljana, Slovenia # CNR Institute of Biostructures and Bioimaging, Catania, Italy @ Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, and National Institute for Biostructures and Biosystems, Section of Catania, Catania, Italy * S Supporting Information ABSTRACT: The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms. T he voltage-dependent anion channel (VDAC) is a 30 kDa pore-forming protein mainly located in the outer mitochondrial membrane (OMM). Its main function is to form a general pore for ions and small metabolites, among which the energetic nucleotides ATP, ADP, and NADH are particularly important. VDAC is anion selective in the open state, but it switches to a partially closed state when the applied transmembrane voltage is increased above 30−40 mV, with the closed state characterized by a reduced permeability and slight cation selectivity. 1−4 In addition to guaranteeing the exchange of ions and metabolites between the cytosol and the mitochondrion, VDAC has been found to actively interact with other proteins like hexokinase 5 and Bcl-2 family members, 6 playing a fundamental role in the regulation of pathways related to apoptosis 7,8 and cancer. 9,10 Three different VDAC isoforms are expressed in higher eukaryotes. Among the three, VDAC1 is the most abundant, being 10 and 100 times more prevalent than VDAC2 and VDAC3, respectively, in the majority of cells. 3,11,12 VDAC is a large transmembrane channel (outer diameter of 4.5 nm, inner diameter of 2.0−2.5 or 3.0 nm with the N- terminal domain inside or outside, respectively, and height of 4 nm) formed by 19 β-strands arranged in a regular antiparallel organization, whereas the parallel pairing of strands 1 and 19 completes the β-barrel. 13−16 Both the N- and C-termini face the mitochondrial intermembrane space. 17 The most evident Received: April 28, 2015 Revised: August 22, 2015 Published: August 24, 2015 Article pubs.acs.org/biochemistry © 2015 American Chemical Society 5646 DOI: 10.1021/acs.biochem.5b00469 Biochemistry 2015, 54, 5646−5656 Downloaded by UNIV DEGLI STUDI DI CAGLIARI on September 15, 2015 | http://pubs.acs.org Publication Date (Web): August 28, 2015 | doi: 10.1021/acs.biochem.5b00469