Kv7.1 is a cardiac voltage-gated potassium channel that underlies the delayed rectifier current (IKS) in the heart. The slow response to membrane depolarization is a hallmark feature of this channel's physiology, yet the mechanistic basis of how voltage promotes the open potassium conducting state is unknown. We focused on previously identified aromatic residues which might couple the pore and voltage-sensing domains (VSDs) by using a chemical tuning approach whereby aromatic residues are modified by serial fluorination. The data show that serial fluorination at one site (F232 on the S4 helix, within the VSD) resulted in a stepwise voltage-gating shift, where each added fluorine atom further biased channel opening to more negative voltages. Mutant-cycle analysis of proximal positively charged amino acids indicates that F232 likely forms a cation-π interaction with K285, a residue at the tip of the S5 segment in the pore domain. Using cryoelectron microscopy, a partial structure of the F232 penta-F-Phe Kv7.1 (KCNQ1) open channel was resolved to 6 Å. The data support a gating mechanism whereby the F232-K285 cation-π interaction represents an intermediate activated state that is broken prior to channel opening.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Ion channels play central roles in biology and human health by catalyzing the transmembrane flow of electrical charge. These proteins are ideal targets for genetic code expansion (GCE) methods because it is feasible to measure ion channel activity from miniscule amounts of protein and to analyze the resulting data via rigorous, established biophysical methods. In an ideal scenario, the encoding of synthetic, noncanonical amino acids via GCE allows the experimenter to ask questions inaccessible to traditional methods. For this reason, GCE has been successfully applied to a variety of ligand- and voltage-gated channels wherein extensive structural, functional, and pharmacological data exist. Here, we provide a comprehensive summary of GCE as applied to ion channels. We begin with an overview of the methods used to encode noncanonical amino acids in channels and then describe mechanistic studies wherein GCE was used for photochemistry (cross-linking; caged amino acids) and atomic mutagenesis (isosteric manipulation of charge and aromaticity; backbone mutation). Lastly, we cover recent advances in the encoding of fluorescent amino acids for the real-time study of protein conformational dynamics.

Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.