70 M. Priest and F. Bezanilla
4 Outlook
Functional site-directed fluorometry is a powerful tool for investigating in vivo con-
formational dynamics of proteins in real-time. In 18 years it has expanded from
a single canonical voltage-gated potassium channel to a diverse range of VGICs,
LGICs, and transporters. It has been performed with two-electrode, cut-open, and
patch-clamp electrophysiological techniques. It has expanded to reconstituted puri-
fied proteins and even to the single molecule level (Blunck et al. 2008 ). Variations
on the technique have used fluorescent dyes to measure external accessibility of
the Shaker transmembrane domains (Gandhi et al. 2003 ), electric field strength at
different points near the S4 (Asamoah et al. 2003 ), and the rotational mobility of
the dye using fluorescence polarization (Raghuraman et al. 2014 ). Furthermore, by
labeling sites with more than one dye, fluorescence resonance energy transfer has
been accomplished; examples include FRET between TRPV1 turrets upon heating
and application of divalent cations (Yang et al. 2010 , 2014 ), between subunits of
the mechanosensitive ion channel MscL (Corry et al. 2005 , 2010 ; Wang et al. 2014 )
and subunits of gramicidin (Borisenko et al. 2003 ; Harms et al. 2003 ), and within
the ryanodine receptor RyR1 (Fessenden and Mahalingam 2013 ). With the constant
improvement of light recording techniques, the variety of new methods to interpret
the fluorescence changes, and the recent combination of site-directed fluorometry
with unnatural amino acids (Kalstrup and Blunck 2013 ), this technique is expected
to provide a wealth of new information on conformational dynamics with good time
resolution in functioning proteins.
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