Computational protocol: Distinct domains of the β1-subunit cytosolic N terminus control surface expression and functional properties of large-conductance calcium-activated potassium (BK) channels

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Protocol publication

[…] Secondary structure of the wild-type β1-subunit was predicted by submitting its protein sequence to the JPred online tool (). Amphipathicity and potential helical secondary structure on the N-terminal region was analyzed with Heliquest (). The initial tertiary structure of the first 50 N-terminal residues of the wild-type β1-subunit was modeled using the I-Tasser web server (). A number of probable models were built by the server based on multiple threading alignments followed by iterative assemblies of template fragments. The structure with the highest confidence score was next chosen to be further refined using the Swiss-PdbViewer Version 4.1 (). Briefly, the last 10 residues of the modeled peptide were first trimmed to better reflect the predicted and reported consensus secondary structure before several rounds of side-chain fixations and energy minimization were applied sequentially until no further improvement could be achieved. Quality of the enhanced structure was assessed on both the WHAT-IF () and SWISS-MODEL structural assessment () web servers. This predicted wild-type model was then used as a template in the homology modeling of the mutated N-terminal β1 peptides using the Multiple Mapping Method (MMM) web server (). MMM combines the best alternatively aligned fragments along the peptide into a final alignment used for the model construction. All the predicted mutant models were further refined and assessed as above. The rotational and translational orientation of each of the modeled peptide with respect to membrane lipid bilayers was next calculated using the PPM web server (). The peptide/membrane molecular system was subsequently assembled with the lipid replacement method, equilibrated, and minimal dynamics-simulated in a synthetic environment of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers using the CHARM-GUI web server (). The 3D models were visualized using the program Chimera (). […]

Pipeline specifications

Software tools JPred, HELIQUEST, I-TASSER, Swiss-PdbViewer, MMM
Applications Protein structure analysis, Membrane protein analysis
Chemicals Calcium, Potassium