C-type inactivation of K + channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K + channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K + or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K + channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid D-Alanine. Protein semisynthesis was used to introduce D-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K + channel K v AP. For semisynthesis of the K v AP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and K v AP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.
Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semi-synthesis of integral membrane proteins in which solid phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a sumo-fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane spanning peptides required for the semi-synthesis. We demonstrate the feasibility of the modular approach by the semi-synthesis of full-length KcsA K+ channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the iso-steric analog, β-(3-benzothienyl)-L-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K+ conduction and selectivity of the 3BT mutant channels are similar to the wild type, but the mutant channels show a three-fold increase in Rb+ conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K+ conduction over Rb+ conduction.
In this contribution, we report in vitro folding of the archaebacterial voltage gated K+ channel, KvAP. We show that in vitro folding of the KvAP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature and almost quantitative refolding of the KvAP channel is observed at 80 °C. In order to differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the KvAP channel provides a good model system to investigate the folding of a multi-domain integral membrane protein.
Excessive mitochondrial matrix Ca2+ and oxidative stress leads to the opening of a high‐conductance channel of the inner mitochondrial membrane referred to as the mitochondrial permeability transition pore (mtPTP). Because mtPTP opening can lead to cell death under diverse pathophysiological conditions, inhibitors of mtPTP are potential therapeutics for various human diseases. High throughput screening efforts led to the identification of a 3‐carboxamide‐5‐phenol‐isoxazole compounds as mtPTP inhibitors. While they showed nanomolar potency against mtPTP, they exhibited poor plasma stability, precluding their use in in vivo studies. Herein, we describe a series of structurally related analogues in which the core isoxazole was replaced with a triazole, which resulted in an improvement in plasma stability. These analogues were readily generated using the copper‐catalyzed “click chemistry”. One analogue, N‐(5‐chloro‐2‐methylphenyl)‐1‐(4‐fluoro‐3‐hydroxyphenyl)‐1H‐1,2,3‐triazole‐4‐carboxamide (TR001), was efficacious in a zebrafish model of muscular dystrophy that results from mtPTP dysfunction whereas the isoxazole isostere had minimal effect.
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