Biology Reference
In-Depth Information
multicolor, single-molecule fluorescence studies of the structural and compositional dy-
namics of macromolecular complexes involved in signal transduction. We present a gen-
eral, simple, and robust method for stoichiometric, site-specific fluorescence labeling of
expressed GPCRs. The method is based on bioorthogonal conjugation of a fluorescent
reporter group to a genetically encoded azido group introduced into expressed GPCRs
using amber codon suppression.We then present a strategy to reconstitute labeledGPCRs
in native-like membranes and to tether-oriented samples onto surfaces amenable for in-
terrogation by total internal reflectance fluorescence (TIRF) spectroscopy. We describe
how to assemble an automated four-color epifluorescence microscope with SMD-TIRF
optics. Finally, we discuss how to adapt engineered samples for high-throughput imaging
with the aim of understanding the kinetic relationships between ligand binding and the
dynamic regulation of the GPCR signalosome.
15.1
PURPOSE
The protocols described in this chapter provide a foundation for automated, multicolor,
single-molecule fluorescence studies of the structural and compositional dynamics of
macromolecular complexes involved in signal transduction. In particular, we are inter-
ested in the dynamic assembly of signaling complexes (“signalosomes”) involving G
protein-coupled receptors (GPCRs). We present a general, simple, and robust method
for stoichiometric, site-specific fluorescence labeling of GPCRs. The method is based
on bioorthogonal conjugation of a fluorescent reporter group to a genetically encoded
azido group. Furthermore, we describe high-throughput adapted approaches to single-
molecule imaging by total internal reflection fluorescence (TIRF) microscopy.
15.2
THEORY
Single-molecule detection (SMD) fluorescence methods have appeared first in the late
1980s, and there has been almost exponential growth (
Joo, Balci, Ishitsuka,
Buranachai, & Ha
,
2008
) of work in this field, ever since. One of the fascinating
features of these methods is that detailed kinetic information is accessible from
observations of a fluctuating system under equilibrium conditions (
Weiss, 1999
).
Protein-nucleic acid interactions have become the spearhead of biological applications
(
Roy, Hohng, & Ha, 2008
), in part due to favorable kinetics (
Blanchard, 2009
).
The protocols presented in this chapter address a series of technical challenges in
the study of membrane proteins, especially GPCRs.
15.2.1
Site-specific labeling of GPCRs with fluorophores
suitable for SMD-TIRF
Using our amber codon suppression technology (
Ye et al.
,
2008
), it is possible to ge-
netically encode a reactive
p
-azido-phenylalanine (azF) residue with just a single-site
mutation (
Naganathan, Ye, Sakmar, & Huber
,
2013; Ye, Huber, Vogel, & Sakmar
,
