McDermott, A. E. | bioRxiv Science

bioRxiv2019

Stability of Nitroxide Biradical TOTAPOL in Biological Samples

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

biophysics↗

bioRxiv2019

Allosteric studies of an open pH gate KcsA mutant in its activated and inactivated states

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the mean open time of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that has the pH gate open across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about 4 orders of magnitude weaker than that of wild type KcsA at neutral pH and is comparable to the value for wild type KcsA at low pH (pH [~] 3.5). This result strongly supports our assertion that the open pH gate allosterically effects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.\n\nSignificance statementInactivation of potassium channels controls mean open times and provides exquisite control over biological processes. In the highly conserved C-type inactivation process, opening of the activation gate causes subsequent inactivation. We test whether the open state of the channel simply has a poor ability to bind the K+ ion. Previously, activated and inactivated states were stabilized using truncations or a significant pH drop. Here, we use the H25R/E118A constitutively open mutant of KcsA and also observe a large drop in potassium binding affinity. This provides strong evidence that channel opening causes an allosteric loss of ion affinity, and that the central feature of this universal channel inactivation process is loss of ion affinity at the selectivity filter.

biophysics↗