Tertiary and Quaternary Structure Formation of Voltage-Gated Potassium Channels

Voltage-gated potassium channels are ubiquitous and critical for life. They must fold and assemble correctly and target to appropriate sites in the plasma membrane. Failure to do so can lead to inappropriate targeting or function and to pathology. The methods described here were developed to assess in which compartment tertiary and quaternary structure acquisition occurs. The experimental strategies involve identifying quaternary and tertiary interfaces, engineering a pair of cysteines into a cysteine-free voltage-gated potassium channel protein, using bifunctional crosslinking agents, and using an assay of the crosslinked products to determine folding/assembly events. A biogenic intermediate (i.e., nascent chain attached to transfer RNA and the ribosome) is used to probe events inside and at the exit port of the ribosomal tunnel.