Significance Ion channels exist in all organisms. Here, we connect the folding of potassium channel monomers to the kinetics of tetramerization. Rather than adopting a native-like conformation once inserted into the bilayer, monomers initially exist as a structurally heterogeneous ensemble in a protein-dense region. This early clustering of monomers may be a general phenomenon that assists in the assembly of multimeric membrane proteins by pre-localizing the subunits. Folding can occur along fast or slow (misfolded) pathways that can be modulated with mutations that trap monomers in a native-like state. In spite of its name, the C-terminal “tetramerization” domain in KcsA does not enhance tetramerization, suggesting it may play another role in channel function.

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

Engineered antibody fragments (Fabs) have made major impacts on structural biology research, particularly to aid structural determination of membrane proteins. Nonetheless, Fabs generated by traditional monoclonal technology suffer from challenges of routine production and storage. Starting from the known IgG paratopes of an antibody that binds to the “turret loop” of the KcsA K+ channel, we engineered a synthetic Fab (sFab) based upon the highly stable Herceptin Fab scaffold, which can be recombinantly expressed in Escherichia coli and purified with single-step affinity chromatography. This synthetic Fab was used as a crystallization chaperone to obtain crystals of the KcsA channel that diffracted to a resolution comparable to that from the parent Fab. Furthermore, we show that the turret loop can be grafted into the unrelated voltage-gated Kv1.2–Kv2.1 channel and still strongly bind the engineered sFab, in support of the loop grafting strategy. Macroscopic electrophysiology recordings show that the sFab affects the activation and conductance of the chimeric voltage-gated channel. These results suggest that straightforward engineering of antibodies using recombinant formats can facilitate the rapid and scalable production of Fabs as structural biology tools and functional probes. The impact of this approach is expanded significantly based on the potential portability of the turret loop to a myriad of other K+ channels.

assess the significance of toxin residues. Here, we show that geneticallyencoded, membrane-tethered toxins (T-toxins) allow rapid screening of the residues to determine the mechanistic basis for toxin-ion channel interaction, including kinetics parameters of interaction, through study of two channels, KcsA and Kv1.3. First, the structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. Then, T-HmK residues are scanned by point mutation to identify the seven residues in close contact with the KcsA pore. T-HmK-Lys 22 is shown to interact with K þ ions traversing the permeation pathway from the cytoplasm conferring voltage-dependence to the toxin offrate, a classic mechanism we observe as well for HmK peptide with both KcsA and K V 1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and K V 1.3, respectively, via an arginine rather than the canonical lysine, when tethered as well as free peptides. Our study changes a long-held conclusion about the voltage-dependent mechanism of ShK-Kv1.3 interaction, and demonstrates that there are two orientations for SAK1 toxins in K þ channel pores.