Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are poorly understood. In the K+ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K+ channels are associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K+ channels, which provides a foundation for an improved understanding of eukaryotic K+ channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K+ channels.

Proteins display a wealth of dynamical motions that can be probed using both experiments and simulations. We present an approach to integrate side-chain NMR relaxation measurements with molecular dynamics simulations to study the structure and dynamics of these motions. The approach, which we term ABSURDer (average block selection using relaxation data with entropy restraints), can be used to find a set of trajectories that are in agreement with relaxation measurements. We apply the method to deuterium relaxation measurements in T4 lysozyme and show how it can be used to integrate the accuracy of the NMR measurements with the molecular models of protein dynamics afforded by the simulations. We show how fitting of dynamic quantities leads to improved agreement with static properties and highlight areas needed for further improvements of the approach.

The conformational heterogeneity of a folded protein can affect not only its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here, we have examined whether changed native-state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps–ns dynamics of amide groups in the backbone and the methyl groups in the side chains with molecular dynamics simulations to quantify the native-state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: a reduction in conformational dynamics (and entropy estimated from this) of the native state of the L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native-state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

Proteins display a wealth of dynamical motions that can be probed using both experiments and simulations. We present an approach to integrate side chain NMR relaxation measurements with molecular dynamics simulations to study the structure and dynamics of these motions. The approach, which we term ABSURDer (Average Block Selection Using Relaxation Data with Entropy Restraints) can be used to find a set of trajectories that are in agreement with relaxation measurements. We apply the method to deuterium relaxation measurements in T4 lysozyme, and show how it can be used to integrate the accuracy of the NMR measurements with the molecular models of protein dynamics afforded by the simulations. We show how fitting of dynamic quantities leads to improved agreement with static properties, and highlight areas needed for further improvements of the approach.