Biology Reference
In-Depth Information
A
1
D4 sepharose beads
1. DIBO or DBCO
Solubilization
2. Specific elution
azF tagged-Rho in DM
Labeled Rho
HEK293-F
mammalian call
suspension culture
Immunopurified azF tagged-Rho
B
x: Rho residue mutated to azF
1: DIBO
Alexa: Alexa488/555/594/647
N
N
N
3
N
Alexa
O
O
R
1
R
1
N
H
N
H
Alexa
R
2
R
2
x
1
FIGURE 15.1
Labeling scheme of rhodopsin with strain-promoted alkyne-azide cyclooctyne (SpAAC).
(A) Labeling scheme of rhodopsin with strain-promoted alkyne-azide cyclooctyne. First
p-azido-
L
-phenylalanine (azF) was incorporated into opsin heterologously expressed in
HEK293-F suspension cells using amber codon suppression technology. The cells
expressing opsin were harvested and regenerated with 11-cis-retinal. The regenerated cells
were solubilized and immunoprecipitated with 1D4 sepharose 2B resin. After the labeling
reaction, the unreacted dyes were washed away. The labeled receptor was eluted and
recovered in the elution buffer. (B) The dibenzocyclooctyne (DIBO) reagents derivatized with
fluorophore were used to generate fluorescently labeled receptor.
2009
). The site-specific azido group is then conjugated to a fluorophore or any other
biophysical probe (
Fig. 15.1
). The goal here is to introduce a unique amino acid side
chain at a site that would not affect function, but would be informative with respect to
receptor conformational changes related to structural dynamics of signalosomes. Our
method circumvents the fundamental problem in generating site-specific fluorescently
labeled proteins based on single-accessible cysteine mutants, that is, the necessity to
generate first a cysteine-free protein background by identification of nonessential and
accessible cysteines and substitution with alternative amino acids. The problem here is
circular, as one would need a sufficiently informative functional assay to detect any
unwanted side effects of these mutations, while the purpose of the labeling experiment
itself is to establish such an assay.
15.2.2
Single-molecule immunoprecipitation of GPCRs
TIRF methods (
Axelrod, Burghardt, & Thompson, 1984
) allow selective excitation of
fluorophores in the evanescent wave penetrating about 100 nm deep into the aqueous
layer beyond the glass-water interface. Capturing a molecule of interest in this spatial
