Functional Site-Directed Fluorometry 69
fluorophore fluorescence to comprise the total fluorescence change observed during
channel activation (Loots and Isacoff 2000 ; Sørensen et al. 2000 ).
Several strategies have been used to overcome the difficulty of interpreting fluo-
rometry data. One is to accomplish global fluorescent measurements from dozens of
sites, which to date has only been performed in the Shaker voltage-gated potassium
channel. By holistically examining the data, it was seen that S4 moves the most
and S1 the least, that S4 moves relative to S3, that the movement of the pore during
channel opening can be measured from particular sites, and that the S4 also moves
with respect to the rest of the protein during slow inactivation (Gandhi et al. 2000 ;
Pathak et al. 2007 ).
Another strategy is to determine the residues responsible for producing the
changes in fluorescence. For example, in Shaker the S3-S4 linker contains a string
of glutamates, an amino acid known to quench TMR; these residues seem to be the
major component of the fluorescence signal of TMR conjugated to the extracellular
side of the S4 (Blunck et al. 2004 ; Cha and Bezanilla, 1997 ; Sørensen et al. 2000 ).
Another very good example is provided by Pantazis et al. 2010 who demonstrated
that in the BK channel the voltage-dependent fluorescence changes of a dye con-
jugated to a site at the S0 segment were produced by a particular tryptophan at the
extracellular side of the S4. Building on this work, S1 and S2 were seen to undergo
a similar motion to S0, and by tryptophan insertion, S1 was shown to move closer
to S2 upon depolarization (Pantazis and Olcese 2012 ). In this way, particular inter-
actions between the fluorophore and an amino acid or amino acids on the protein
can be defined. With enough granularity, this will allow site-directed fluorometry to
successfully provide insight into structural dynamics, although the bulky size of the
fluorescent dyes may limit its spatial resolution.
A more analytical strategy to interpret fluorescence changes is to propose a ki-
netic model of the conformational change and assign quenching levels to the states,
as was shown to fit a complicated set of biphasic signals observed in constructs of
Shaker with shortened S3-S4 linkers (Sorensen et al. 2000 ). This procedure has
been generalized to a full model of the conformational landscape using the Q-ma-
trix approach (Andrew Plested, personal communication).
Another limitation has been the inability of site-directed fluorometry to easily
provide information on residues towards the intracellular side of the protein or to
inaccessible sites in the transmembrane regions. Recently, this limitation has been
surmounted using unnatural amino acids (Kalstrup and Blunck 2013 ), which are
discussed in more detail in chapter Incorporation of Non-Canonical Amino Acids of
this book. Alternatively, fluorometry performed on purified proteins reconstituted
into artificial bilayers can provide labeling access to intracellular residues (Blunck
et al. 2008 ). Yet another option for intracellular access is patch-clamp fluorometry
which takes advantage of an inside-out configuration to obtain dye accessibility
(Zheng and Zagotta 2000 ).