Functional Site-Directed Fluorometry 57
to the development of site-directed fluorometry, examination of conformational
changes in membrane proteins was possible with a diverse range of techniques,
including electron microscopy (Unger and Schertler 1995 ; Unwin 1995 ), atomic
force microscopy (Butt et al. 1990 ; Müller et al. 1995 ), Fourier transform infrared
difference spectroscopy (FTIR) (Sonar et al. 1994 ; Souvignier and Gerwert 1992 ),
site-directed spin labeling for electron paramagnetic resonance (EPR) (Farahbakhsh
et al. 1993 ; Steinhoff et al. 1994 ), fluorescence spectroscopy (Dunn et al. 1980 ;
Gether et al. 1995 ), and accessibility studies (Slatin et al. 1994 ; Yang and Horn
1995 ). However, each of these techniques suffered from limitations that functional
site-directed fluorometry was able to surmount.
Electron microscopy and atomic force microscopy suffer from a lack of temporal
resolution; while steady-state conformations can be investigated little information
is provided regarding the transition between these states. Furthermore, voltage-
driven conformational changes remain exceedingly difficult to measure by more
purely structural methods such as atomic force microscopy or electron microscopy.
Finally, these techniques required either membrane proteins tractable to purifica-
tion and reconstitution (Unger and Schertler 1995 ), or were limited to a very select
group of membrane proteins found in high levels in appropriate membranes, such as
the nicotinic acetylcholine receptor of the Torpedo ray postsynaptic membrane (Un-
win 1995 ) or the bacteriorhodopsin of the purple membrane (Müller et al. 1995 ).
EPR and FTIR had achieved temporal resolution on the 10–100 μs time-scale, re-
spectively (Souvignier and Gerwert 1992 ; Steinhoff et al. 1994 ), but were similarly
largely limited at the time to bacteriorhodopsin, and were incapable of measuring
voltage-driven conformational changes. Fluorescence spectroscopy was limited by
the same issues, with experiments taking place in nAChR-rich membranes (Dunn
et al. 1980 ) or in purified proteins reconstituted into liposomes (Gether et al. 1995 ).
Additionally, for all of these techniques simultaneous functional assays of activity
remain challenging.
Accessibility studies, on the other hand, had demonstrated many of the capa-
bilities of site-directed fluorometry. They could be applied to a chosen ion channel
expressed in an in vivo preparation and held under voltage-clamp with changes
observed on a 10 μs time-scale, and accessibility could be tested at multiple cho-
sen sites within the protein of interest and correlated with electrophysiological re-
cordings (Yang and Horn 1995 ; Yang et al. 1996 ). Accessibility studies suffered
from two limitations in comparison with site-directed fluorometry. One was that
the information provided was intrinsically limited to whether a particular site in
the protein was accessible to the extracellular or intracellular space under different
conditions. The second was that in order for accessibility to be measured, the probes
needed to significantly perturb the function of the channel in order for the effect to
be measured. In contrast, the addition of fluorophores to an ion channel or other
membrane protein frequently produces minimal changes in the functional activity
of the protein (Cha and Bezanilla 1997 ; Mannuzzu et al. 1996 ).
With its development, functional site-directed fluorometry provided a technique
that combined the strengths of cysteine accessibility studies ‒ studying a chosen
membrane protein of interest in vivo, in an animal cell membrane under voltage-