Structural Dynamics of an Isolated Voltage-Sensor Domain in a Lipid Bilayer  Sudha Chakrapani, Luis G. Cuello, D. Marien Cortes, Eduardo Perozo  Structure  Volume 16, Issue 3, Pages 398-409 (March 2008) DOI: 10.1016/j.str.2007.12.015 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Three-Dimensional Topology and the Oligomeric State of the Isolated VSD of KvAP (A) A schematic representation of the full-length KvAP channel showing the location of the VSD and the PD (left). Ribbon representation of the TM helices as depicted by the crystal structure of the isolated VSD (right). (B) Gel-filtration chromatography of the isolated VSD shows a dimeric configuration in n-decyl-β-D-maltopyranoside (red) and a monomeric state in n-octyl-β-D-glucopyranoside (black). (C) FRET-based assay of the aggregation state of the isolated VSD and the FL channel (FC) in asolectin and in a mixture POPC:POPG. Mutant A111C was used to colabel the protein with rhodamine and fluorescein. A prominent rhodamine peak at 565 nm is an indication of protein aggregation on the membrane. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 EPR-Based Structural Analysis of the Isolated VSD (A) Mobility ΔHo−1 (black), O2-accessibility ΠO2 (red), and NiEdda-accessibility ΠNiEdda (blue). The gray regions represent the limits of the TM segments. (B) The average mobility and accessibility parameters calculated for each of the TM segments. Each box denotes an individual TM segment, with SDs marked as error bars. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Comparison of Residue-Specific Environmental Parameters in the FL Channel and Isolated VSD (A) An overlap of representative X-band continuous-wave EPR spectra of spin-labeled mutants from S1 and S4 in the FL channel (black) and isolated VSD (red). The black arrow shows the location of the immobile component of the spectra. (B) Profile of changes in the ΠO2 parameters. The values were normalized to the maximum ΠO2 measured within each data set. The blue arrows mark the break in the α-helical periodicity of the accessibility. Positions of the gating charges in S4 are highlighted by yellow ovals. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Frequency and Vectorial Analysis of Environmental Parameters The experimentally determined EPR parameters were mapped onto the crystal structure of the isolated VSD (Protein Data Bank [PDB] code: 1ORS). ΠO2 is shown in red (A and B) and ΠNiEdda in blue (C). The accessibility parameters are plotted in polar coordinates such that the sum vectors point toward the face of the helix with highest mobility (black) or accessibility (ΠO2, red; ΠNiEdda, blue) values. The solid arrows denote the isolated VSD while the broken arrows represent the FL channel. The direction of the calculated moments in the upper and lower halves of the S3 and S4 segment shows a break in the helices accompanied by a twisting of the solvent-exposed surfaces. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 Aqueous Crevices within the VSD (A) A plot of the normalized ΠNiEdda values of the residues in the S3 segment in the isolated VSD (blue) and FL channel (black). (B) ΠNiEdda values mapped onto the S3 segment to show the depth of water-accessible areas (blue arrows) within the transmembrane region of the protein. The dotted lines denote the putative limits of the membrane. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 6 Mapping the Molecular Interacting Surface of the VSD with the PD Fractional differences in the ΔHo−1 and ΠO2 measured for the corresponding residues in the isolated VSD and in the FL channel mapped onto the sensor structure and color coded with a gradient from red to white to blue. Red denotes increase and blue represents decrease in the environmental parameter. The dotted black lines mark the surface of highest impact. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 7 Evaluation of the Open-State Structure of the Kv Channels (A) The delta values were mapped onto the coordinates of the Kv1.2-Kv2.1 chimera structure (PDB code: 2R9R). (B) Model for the open inactivated state of KvAP based on EPR measurements. The top panel shows the view from the intracellular part of the channel. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 8 Comparison of the Sequence Variability within the VSD in Eukaryotic Kv Channels, Hv Channels, and the VSP Family Sequence alignment of the TM segments comprising the VSD (top). The filled star marks the position of the break in S3. An overlap of the calculated sequence variability at each position (gray background) and the corresponding normalized ΠO2 measured within the FL channel (blue) and isolated VSD (red) of KvAP. Positions corresponding to charge residues are marked by arrows. Highly impacted residues in perturbation analyses in eukaryotic Kv channels are shown in orange circles. Structure 2008 16, 398-409DOI: (10.1016/j.str.2007.12.015) Copyright © 2008 Elsevier Ltd Terms and Conditions