56 M. Priest and F. Bezanilla
1 Introduction
Fluorescence has long been used in biology to follow molecular motions in proteins.
As fluorescence intensity of many fluorophores depends on environmental factors,
such as collisional quenching, anisotropy, or hydrophobicity, it is then possible to
infer conformational changes near a fluorophore. Of the three amino acids that are
fluorescent, only tryptophan has a large enough extinction coefficient to be of prac-
tical use. However, there are several limitations to measuring this endogenous fluo-
rescent signal. In a protein that has multiple tryptophan residues, mutating until a
single tryptophan remains is often unfeasible and the interpretation of changes in
tryptophan fluorescence during function becomes difficult. In addition, as trypto-
phan absorbs in the UV, fluorescence measurements in live cells become extremely
difficult due to absorption and emission of many intrinsic fluorophores. A possible
solution to the problems posed by intrinsic fluorophores is to insert a fluorescent
probe in a specific site of the protein and then measure its fluorescence while the
protein is functioning. In this case, the fluorophore is selected to absorb and emit
in the visible or infrared part of the spectrum and to have specific properties, such
as hydrophobic/hydrophilic sensitivity, electric field sensitivity, pH sensitivity, etc.
This technique applied to membrane proteins that are under voltage clamp condi-
tions has been called voltage clamp fluorometry. A more accurate name for this
technique is functional site-directed fluorometry because it records fluorescence in
specific sites while monitoring function at the same time.
Functional site-directed fluorometry measures the fluorescence changes of a dye
conjugated to a specific residue on an ion channel or other membrane protein as it
moves in response to an applied change in voltage or a change in some other modu-
lator of conformational state. Although FRET applications have been developed,
here we focus on applications of site-directed fluorometry in which a single dye is
attached to a single site on the protein. Initial studies using site-directed fluorometry
studied the conformational changes of the Shaker voltage-gated potassium chan-
nel (Cha and Bezanilla 1997 ; Mannuzzu et al. 1996 ). Rather than simply examin-
ing ionic currents (Timpe et al. 1988 ) or gating currents (Armstrong and Bezanilla
1973 ) as indirect readouts of conformational changes of the channel, site-directed
fluorometry provided for the first time the ability to directly view local conforma-
tional changes of a channel protein in real-time. The dynamics of these changes
could be directly measured by correlating the changes in fluorescence of a dye con-
jugated to the protein with known transitions between conformational states. This
ability of site-directed fluorometry to observe conformational dynamics is a major
strength of the technique.
Site-directed fluorometry arose specifically out of a desire to answer the question
of whether the S4 segment in the voltage-gated potassium channel, suggested to be
the voltage sensor of the channel, moves when the gating charges move in response
to changes in membrane potential. More generally, the technique provided a wholly
novel ability to access real-time changes of protein conformation and to examine
the transitions between states rather than simply the nature of the end states. Prior