4 S. A. Pless and C. A. Ahern
recent extension of the basic idea of cysteine modification is described in chapter
Bioreactive Tethers. Here, the authors describe how bifunctional tethers can be
used to probe ion channel structure and function. The basic principle is that one
end of the tether can be linked to the channel domain of interest via a (cysteine-
reactive) thiol group, while the other contains a non-covalent targeting moiety,
such as a small molecule, peptide, or even small proteins targeting an ion channel.
This approach results in much-enhanced avidity of these molecules towards their
target and has been essential in elegant studies to probe subunit stoichiometry.
Chapter Flipping the Photoswitch: Ion Channels Under Light Control focuses on
a closely related version of the bioreactive tether principle, in which the tether
contains a synthetic photochrome that can be used to optically control the avail-
ability of a ligand or crosslinker. Thanks to the rapid and reversible conformation-
al conversion of these “photoswitches”, light-dependent ion channel agonists, an-
tagonist or modulators can now be used both in in vitro and in vivo settings. While
the proceeding chapters have relied on changing the protein sequence within the
bounds of the genetic code and therefore focused on the modification of natu-
rally occurring amino acids, either by protons (chapter Engineered Ionizable Side
Chains) or cysteine-reactive moieties (chapters Cysteine Modification: Probing
Channel Structure, Function and Conformational Change, Functional Site-Di-
rected Fluorometry, Bioreactive Tethers and Flipping the Photoswitch: Ion Chan-
nels Under Light Control), chapter Incorporation of Non-Canonical Amino Acids
introduces the concept of an expanded genetic code and its nascent application to
ion channel and receptor proteins in biochemical and cellular environments. By
recoding one of the naturally occurring stop codons (most commonly the TAG or
amber stop codon), this technique allows to introduce unnatural amino acids into
a protein of interest. As these changes are genetically encoded, probes can be in-
serted anywhere into the protein. This can serve to introduce subtle variations of
naturally occurring amino acids or to endow the protein of interest with entirely
new functionalities, such as side chains that are fluorescent and can act as (photo)
crosslinkers. This chapter also covers an alternative way to introduce unnatural
amino acids by way of ligating a synthetically made peptide (containing an UAA)
with a heterologously expressed polypeptide.
Over the recent decade, many of the above approaches have seen a period of
unprecedented expansion and the future will no doubt continue to bring on many
exciting approaches at the interface of chemistry and biology to allow us to dis-
sect the atomic basis for ion channel function and physiology with ever increasing
precision.