Unnatural amino acid mutagenesis for site-specific incorporation of keto and azido
functionalities into G protein-coupled receptors
Shixin Ye, Thomas Huber, Amy Grunbeck, Thomas P. Sakmar
Laboratory of Molecular Biology & Biochemistry, The Rockefeller University, New York, NY 10065
ABSTRACT:
Results
Introduction: The insertion of unnatural amino acids into proteins using amber stop
codon suppression has shown promise as a technique for probing protein
structures. To investigate applications to studies of G protein-coupled receptors,
we have developed methods that allow incorporation of each of three tyrosine
analogues –– p-acetyl-phenylalanine (Acp), p-benzoyl-phenylalanine (Bzp), and
p-azido-phenylalanine (Azp) – into GPCRs site-specifically (1) at high yields in
mammalian cell culture. The unique keto and azido functionalities allow specific
attachment of tags and fluorophores into GPCRs by hydrazone (2) and
Staudinger-Bertozzi ligation (3) respectively under physiological
conditions. Together with cysteine-specific labeling methods, the hydrazone and
Staudinger-Bertozzi ligations will make it possible to introduce pairs of
fluorophores into GPCRs in a general way. The benzoylphenone moiety generates
a reactive species by irradiation with UV light, which would covalently crosslink
to any nearby protein. Therefore, the binding interface of GPCR and its G protein
can be systematically mapped.
CONCLUSIONS (chemical labeling):
(A)
(A) Purified luciferase (wt and
Y70Acp mutant) on Ni-NTA
(left panel) or 1D4 resins (right
panel) was reacted with 1mM
biocytin-hydrazide. The eluents
were analyzed by western
blotting.
(B)
(B) Purified luciferase (wt and Y70Acp mutant) on
Ni-NTA resins was reacted with biocytin-hydrazide
at various concentration (left panel) for 24hr.
Purified luciferae (wt and Y70Azp mutant) on NiNTA resins was reacted with biotin-phosphine at
0.1mM for 24hr (right panel). The eluents were
analyzed by western blotting.
Amber stop codon suppression in GPCRs
a. General scheme
(C)
(C) Purified luciferase (wt and
Y70Acp mutant) on 1D4 resins
was reacted with biocytinhydrazide or biotin-PEO4-HNAA
at various concentrations. The
eluents were analyzed by
western blotting.
Table 1.
Using luciferase and rhodopsin as model systems, we have
demonstrated that hydrazone and Staudinger-Bertozzi ligations allow
covalent attachment of labels under physiological conditions. Both
proteins are functional after ~12hr reactions. We have also noticed
that wild-type luciferase or rhodopsin in the absence of a keto moiety
reacted with hydrazide and caused nonspecific attachment of labels.
The chemical nature of the observed nonspecific reaction is due to
naturally presence of keto/aldehyde groups in proteins (4). Various
proteins can carry different levels of keto/aldehyde groups (table 1).
The heavier the protein, the higher the chance it gets one keto or
aldehyde group present. This nonspecific labeling reaction has been
overlooked because model proteins being studied for the hydrazone
ligation so far are mainly small molecular weight proteins (5-20kD).
Together with cysteine-specific labeling methods, the hydrazone and
Staudinger-Bertozzi ligations will make it possible to introduce pairs
of fluorophores into GPCRs in a general way. This is a prerequisite
for single molecule fluorescent resonance energy transfer (smFRET)
studies, which will yield receptor dynamic information not readily
available by other experimental methods. Comparisons of these
ligations are discussed in table 2.
Photo-crosslinking with Bzp
(D)
b. Three tyrosine analogues
(A) In vitro labeling of functional rhodopsin mutants
containing Acp at positions 29, 102, or 274. UV-vis
spectra before (solid lines) and after photobleaching
(dashed lines) of rhodopsins treated with fluorescein
hydrazide.
(B) Difference spectra (dark minus light spectra) of
labeled rhodopsin mutants generated from (A).
(C) Stoichiometric ratios of fluorescein/rhodopsin
were determined after normalizing the amount of
rhodopsin based on the absorbance at 500 nm. The
molar extinction coefficients used for calculations
were 42,000 M-1cm-1 for rhodopsin and 93,200
M-1cm-1 for fluorescein. F/R (fluorescein/rhodopsin
molar ratio).
(D) Fluorescein detection by a Typhoon 9400 Image
Scanner using an excitation/emission filter set
optimized for fluorescein.
Chemical Labeling with Acp and Azp
a. Reaction schemes
b. Biotin reagents
(A) Biotin reagents for hydrazone ligation
O
HN
NH
O
H
N
S
NHNH2
O
HN
NH2
Biocytin-hydrazide (Invitrogen)
O
NH
H
N
S
O
O
O
NHNH2
O
O
Biotin-PEO4-hydrazide (Pierce)
O
HN
O
NH
N
H
N
N
H
N
o
o
S
N
o
O
O
Biotin-PEO3-HNAA (Solulink)
(B) Biotin reagents for Staudinger-Bertozzi ligation
O
HN
O
NH
O
H
N
S
H
N
O
O
P
O
O
O
Biotin-PEO3-phosphine
c. Luciferase as a model protein to study the labeling chemistry
N-term
Hisx6
Luciferase
C-term
1D4
Luciferase
1D4
Y70amb
Hisx6
Human IgG (Sigma) as a model protein testing the
presence of naturally occurring hydrazide reactive
groups. IgG at 1mg/ml in 100mM phosphate,
pH=6.0, 150mM NaCl, react with 1mM
fluorescien hydrazide (Invitrogen: C356) in the
absence and in the presence of additives for 24h
incubation, RT. Reaction mixture (200ul) is
purified with Sephadex G50 (5ml bed, 6cm
length). Fractions are collected at 200ul for 20
times. Fractions containing purified IgG are
identified by photometric plate reader
(SpectraMax 250) and pooled for further analysis.
Quantify F/P ratio is done by the 2nd derivativeanalysis of measured spectra: (494nm
fluorescien)/(301nm protein).
Table 2.
Mapping the binding interface between two proteins by the site-directed
mutagenesis with Bzp.
A. The amber stop codon suppression method introduces Bzp at any location
in the putative binding interface.
B. Under UV-light, Bzp moiety generates free radical species that covalently
attaches to a nearby protein.
C. The crosslinked product can be identified by conventional immunoblotting.
D. Systematically introducing Bzp at the binding interface will help to
identify the key amino acids involved in binding.
Establishing the crosslinking procedure in a model GPCR - rhodospin.
Rho-Bzp
mutants
Membrane
prep
biotinylated
Gt peptide
wash
UV light
Crosslinking
SDSPAGE
Western
blotting
anti-1D4 detection:
rhodopsin
(A)
(B)
streptavidin detection:
rhodopsin crosslinked to
the Gt peptide
(A) Crystal structure of opsin/Gt peptide complex (adapted from ref 5).
(B) Crosslinking of Rho-Bzp mutants to the Gt peptide. Rhodopsin mutants
containing Bzp at T229 and T243 positions form covalent bonds with Gt peptide
after photo-crosslinking with UV light. The biotinylated Gt peptide is detected by
HRP-streptatvidin (top panel), and rhodopsin is detected by anti-1D4 antibody
(bottom panel).
CONCLUSIONS (photo-crosslinking):
1. We use rhodopsin as a model system to establish the crosslinking conditions with Bzp. Site-specific incorporation of a benzophenone
moiety into rhodopsin at specific locations (e.g. T229 and T243), followed by irradiation with UV light generates a reactive species that
crosslinks it to the Gt peptide.
2. This method will provide an alternative strategy to map the binding interface of a GPCR and its G protein. Evidence has accumulated that
dimerization plays an important role in the GPCR signaling process. Homodimerization and heterodimerization of GPCRs can modify the
physiological response through interaction or activation of its neighbor.
REFERENCES:
1.
2.
3.
4.
5.
Ye, S.X. et al. J. Biol. Chem. 283: 1525-1533 (2008).
Cornish, V.W., Hahn, K.M., & Schultz, P.G. J. Am. Chem. Soc. 118: 8150-8151 (1996).
Agard, N.J., Baskin, J.M., Prescher, J.A., Lo. A., & Bertozzi, C.R. ACS Chem. Biol. 1: 644-648 (2006).
Ahn, B., Rhee, & S.G., Studtman, E.R. Analytical Biochem. 161: 245-7 (1987).
Scheerer, P. et al. Nature 455: 497-503 (2008).