ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we employ NMR measurements of backbone amide 15N spin relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps-ns timescale, increased linewidths observed for eleven backbone amide 15N resonances identify chemical or conformational exchange contributions to spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane–cytoplasm interface (TM2 gate), and in the cytoplasmic C‐terminal domain (CTD), an amphiphilic four‐helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH‐induced open‐to‐closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl‐protonated at Leu/Val residues to allow observation of both states by NMR, and spin‐labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0–5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin–spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated “closed” channels. We extended these findings for isolated CTD‐derived peptides to full‐length KcsA and have established a contribution of the CTD to KcsA pH‐controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.

The 35-residue ShK peptide binds with high affinity to voltage-gated potassium channels. The dynamics of the binding surface was studied recently with (microsecond to millisecond) (15)N relaxation dispersion and (picosecond to nanosecond) (15)N spin relaxation of the N-H bonds. Relaxation dispersion revealed microsecond conformational-exchange-mediated exposure of the functionally important Y23 side chain to the peptide surface. The spin relaxation parameters acquired at 14.1 and 16.45 T have been subjected to model-free (MF) analysis, which yielded a squared generalized order parameter, S(2), of approximately 0.85 for virtually all of the N-H bonds. Only a "rigid backbone" evaluation could be inferred. We ascribe this limited information to the simplicity of MF in the context of challenging data. To improve the analysis, we apply the slowly relaxing local structure (SRLS) approach, which is a generalization of MF. SRLS describes N-H bond dynamics in ShK in terms of a local potential, u, ranging from 10 to 18.5 kBT, and a local diffusion rate, D2, ranging from 4.2 × 10(8) to 2.4 × 10(10) s(-1). This analysis shows that u is outstandingly strong for Y23 and relatively weak for K22, whereas D2 is slow for Y23 and fast for K22. These observations are relevant functionally because of the key role of the K22-Y23 dyad in ShK binding to potassium channels. The disulfide-bond network exhibits a medium-strength potential and an alternating wave-like D2 pattern. This is indicative of moderate structural restraints and motional plasticity, in support of, although not directly correlated with, the microsecond binding-related conformational exchange process detected previously. Thus, new information on functionally important residues in ShK and its overall conformational stability emerged from the SRLS analysis, as compared with the previous MF-based estimate of backbone dynamics as backbone rigidity.

Membrane-embedded proteins (MPs) are central to a wide range of cellular processes. Despite their importance, structural studies of MPs are hindered by expression difficulties and the need for stabilization in a membranemimicking environment. High-resolution NMR methods can investigate structure and function of MPs due to methodological advances and new membrane-like assemblies for stabilization of MPs. In this perspective of the field, we introduce the challenges and opportunities of NMR studies of membrane proteins, briefly surveying membrane-mimicking systems and their application in structure determination. A case study then focuses on the C-terminal domain of the bacterial potassium channel KcsA, describing how improvements in membrane-mimicking conditions eventually enabled us to present a structural view of the pH-dependent behavior of this cytoplasmic channel domain. The results highlight prerequisites for a successful study of MPs and the potential for future investigations.

The cover picture shows NMR detecting previously unobserved backbone motions of the 35-residue marine potassium channel blocker ShK. By measuring the relaxation of 15 N magnetic moments of ShK (PDB ID: 1roo, shown as a white surface) under different experimental conditions, the existence of a lowly populated conformer was inferred. As described in the article by J. H. Chill et al. on p. 2402 ff., relaxation dispersion curves (below, left) identified amino acids influenced by this "molecular melodrama", thus focusing our attention on the helix-kink-helix motif (red) and, in particular, on residues Lys22 and Tyr23 (blue and magenta, respectively). Increased exposure of Tyr23 in the minor conformer is apparently a key molecular event in ShK blocking of voltage-gated potassium channels (top right); this underscores the potential role of conformation selection in the binding of polypeptidic inhibitors to their biological targets.