Volume 24, Issue 4, Pages 585-594 (April 2016) Molecular Plasticity of the Human Voltage-Dependent Anion Channel Embedded Into a Membrane  Lin Ge, Saskia Villinger, Stefania A. Mari, Karin Giller, Christian Griesinger, Stefan Becker, Daniel J. Müller, Markus Zweckstetter  Structure  Volume 24, Issue 4, Pages 585-594 (April 2016) DOI: 10.1016/j.str.2016.02.012 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 AFM Topographs of hVDAC1 Reconstituted into Lipid Membranes (A) Overview showing membrane patches. (B) High-resolution topography showing the membrane surface peppered with small pores. Patches of densely packed pores are surrounded by higher protruding lipid membrane, which makes it difficult to contour hVDAC1 molecules by the AFM tip. (C) At higher resolution, the individual pores formed by hVDAC1 become better visible (dashed circles). Single-molecule force spectroscopy experiments of hVDAC1 channels were conducted on such densely packed patches. Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Mechanical Unfolding of Membrane-Embedded hVDAC1 (A) Schematics of the single-molecule force experiment. A single hVDAC1 has been non-specifically attached to the tip of an AFM cantilever. Increasing the distance of tip and membrane stretches the polypeptide bridging AFM tip and proteoliposomes. This mechanical stress establishes a force that induces the stepwise unfolding of hVDAC1. (B) Force-distance (FD) curves recorded during unfolding show force peaks (arrowheads) that correspond to interactions established by unfolding intermediates of hVDAC1. Force variations at low distances are due to unspecific interactions, which occur in the contact area of the AFM tip and the membrane. Some FD curves lacked individual force peaks indicating missing unfolding intermediates. Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Unfolding Intermediates and Pathways of hVDAC1 (A) Superimposition of 708 FD curves shows the reproducibility of the FD pattern recorded upon unfolding single hVDAC1 molecules. Colored lines are worm-like chain (WLC) curves fitting individual force peaks. Numbers denote contour lengths (amino acids) of the unfolded polypeptide chain obtained from WLC fits. (B) Probability of detecting unfolding force peaks at certain contour lengths. To obtain the histogram, every force peak of every FD curve of the superposition (n = 708) was fitted with the WLC model and taken into account. The most probable positions ± SD given above each force peak were determined by multiple Gaussian fits. (C) Average contour lengths of force peaks (position outlined by rectangles) locate structural segments of hVDAC1 (equally colored structures) that unfolded within single steps. β strands and polypeptide loops are numbered. For reproducibility of the FD pattern, see also Figure S1. Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Binding of Ca2+ to hVDAC1 (A) Changes in the magnitude of 15N chemical shift deviations (CSD) observed in 1H,15N-TROSY spectra of hVDAC1 induced by a ∼16-fold excess of Ca2+ (black) and Mg2+ (cyan). The horizontal line indicates the average magnitude of the 15N CSD threshold for non-shifting residues. The topology of hVDAC1 is shown on top. (B and C) Residues with a magnitude of 15N CSD larger than 0.05 ppm (orange) and broadening below a ratio of 0.8 (red) in (A) were mapped onto the 3D structure of mVDAC1 (PDB: 3EMN). Residues not observable or not assigned are colored white. (D) Electrostatic potential, which was calculated in the absence of a membrane using DelPhi (Li et al., 2012), is mapped onto the surface of the hVDAC1 structure. A black sphere indicates E73. Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Kinetic, Energetic, and Mechanical Properties of Membrane-Embedded hVDAC1 in the Absence and Presence of Ca2+ or Mg2+ (A) Top view and (B) side view of the 3D structure of hVDAC1 showing the structural segments stabilizing hVDAC1. (C–F) Transition state distance xu (C), free energy barrier height ΔG‡ (D), transition rate k0 (E), and spring constant κ (F) of stable structural segments in the absence (left) and presence of 5 mM Ca2+ (middle) or 5 mM Mg2+ (right). The color of the hVDAC1 backbone roughly indicates the value for each parameter as indicated by the scale bars. Values were taken from Table 1. See also Figure S3. Structure 2016 24, 585-594DOI: (10.1016/j.str.2016.02.012) Copyright © 2016 Elsevier Ltd Terms and Conditions