Introduction 3
in the light of only 20 naturally occurring amino acids it is not always possible to
probe the structural or functional contribution of a side chain in a subtle way, much
less endow the protein with new functionalities. It is this shortcoming of engineer-
ing traditional mutations into ion channels that has sparked an immense interest, as
well as seminal progress in the development of leading edge tools for the application
of diverse chemical and biological techniques, referred to commonly as ‘chemical
biology’ to further study ion channel and transmembrane receptor proteins. It is the
goal of this book to capture the diversity of chemical biology techniques currently
available and to describe their application to model ion channel and transmembrane
receptor systems. Where possible, we have tried to cover different types of ion
channels that have been subjected to a given approach to emphasize the broad ap-
plicability of these techniques.
In chapter Engineered Ionizable Side Chains the authors describe an elegant
approach for the minimal perturbations of ionizable side chains. The binding and
unbinding of protons to and from an engineered side chain causes the charge of
the protein to fluctuate by one elemental unit, and the subsequent changes in
single channel amplitudes can then be used to deduce the proximity of the in-
troduced side chain to the channel pore, while kinetics of binding and unbinding
of protons allow to deduce the side chains pKa value and hence the electrostat-
ics of its microenvironment. Chapter Cysteine Modification: Probing Channel
Structure, Function and Conformational Change lays the foundation for one of
the most widely used approaches in functionalizing ion channels to study their
structure, function and pharmacology by introducing the concept of harnessing
the unique and selective reactivity of cysteine side chains. The fundamental idea
is to probe the reactivity and accessibility of engineered cysteine side chains
within the protein of interest to identify pore-lining residues, (ant)agonist and
modulator binding sites, as well as regions whose conformation changes as pro-
teins transition between different functional states. Further, introducing pairs of
cysteine side chains in order to assay their ability to form disulphide bonds has
proven a very powerful method to monitor changes in proximity and establish
conformational changes within a protein. Furthermore, the fundamental concept
of using the unique reactivity of cysteine side chains has given rise to numerous
approaches, described in chapters Functional Site-Directed Fluorometry, Biore-
active Tethers and Flipping the Photoswitch: Ion Channels Under Light Control,
which allow to introduce new functional groups into the protein of interest by
conjugating them to an engineered cysteine side chain. Chapter Functional Site-
Directed Fluorometry on the voltage-clamp fluorimetry technique explains a now
widely-used variation of cysteine modification that allows the attachment of an
environmentally sensitive fluorescent dye to an introduced cysteine residue. This
permits the simultaneously monitoring of movements at channel gate (through
electrophysiology) and in a domain of interest (through monitoring the quantum
yield of an introduced dye). The approach can therefore provide a direct read out
of protein conformational changes, including even electrophysiologically silent
ones, providing a major advantage in pharmacological studies. Another, more