A photo-cross-linking strategy to map sites of protein-protein interactions

Article abstract of DOI:10.1002/chem.201000441

(Figure Presented) Clicked! Exemplified on a G-proteincoupled receptor (GPCR; see figure)/ G-protein model system, a specific protein-peptide interaction was analyzed by using a G-protein-derived peptide containing p-ethynylbenzoyl-modified phenylalanine. Click chemistry with a biotin propyl azide probe enabled isolation of photo-cross-linked products and facilitated mass spectrometric analysis of the protein-peptide interaction sites.

Full text of DOI:10.1002/chem.201000441

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DOI: 10.1002/chem.201000441
A Photo-Cross-Linking Strategy to Map Sites of Protein–Protein Interactions
Yihui Chen,[b] Yalin Wu,[b] Peter Henklein,[c] Xiaoxu Li,[d] Klaus Peter Hofmann,[a]
Koji Nakanishi,*[b] and Oliver P. Ernst*[a]
Chemical and photochemical cross-linking are potentially
powerful approaches for investigating protein–protein inter-
actions and identification of interaction sites.[1] The ap-
proaches complement high-resolution X-ray diffraction and
NMR spectroscopy methods for three-dimensional protein
structure analysis, especially when proteins are only avail-
able in small amounts or are difficult to crystallize. The
(photo)cross-linked products are typically analyzed by pro-
teolytic digestion and mass spectrometry, yielding, in ideal
cases, sequence and specific amino acid(s) of the interaction
sites. The fast reaction of photo-cross-linking allows its ap-
plication to short-lived protein complexes. To facilitate mass
spectrometric analysis, enrichment of the digested photo-
cross-linked products is desirable. Herein, we describe a
novel photo-cross-linking strategy based on such an enrich-
ment step and capable of mapping sites of protein–protein
interactions. As a test case, we applied the strategy to a
model system represented by the G-protein-coupled recep-
tor (GPCR) rhodopsin and its G protein transducin (Gt).[2]
These two proteins are key enzymes in the visual process
and were chosen previously for (photo)-cross-linking stud-
ies.[3]
GPCRs, also called seven-transmembrane receptors, are
among the most important drug targets and are involved in
many physiological processes by routing extracellular signals
across the cell membrane to different intracellular signaling
pathways.[4] Binding of extracellular ligands causes a confor-
mational change in the GPCR to enable its cytoplasmic
domain to catalyze GDP/GTP exchange in heterotrimeric G
proteins (Gabg); the process that initiates the signaling cas-
cade. Visual signal transduction in the retinal rod cell is a
prototypical sample of G-protein-coupled signaling systems
in which the GPCR rhodopsin with its covalently bound
chromophore 11-cis-retinal acts as a photoreceptor to detect
single photons.[2,5] In its photochemically functional core,
rhodopsin contains the covalently bound ligand 11-cis-reti-
nal, which stabilizes the inactive rhodopsin state. Photon ab-
sorption causes retinal cis!trans isomerization, leading to
in situ formation of an activating ligand in the binding site
and subsequent activation of rhodopsin.
The crystal structures of rhodopsin, as well as that of
opsin, the ligand-free apoprotein, are known.[6] The latter
features a more open conformation of the cytoplasmic
domain, which represents (with respect to G protein cou-
pling) an active GPCR state.[2] For signal transfer to the cog-
nate G protein called transducin (Gt), the C terminus of the
[a] Prof. Dr. K. P. Hofmann, Dr. O. P. Ernst
Institut fꢀr Medizinische Physik und Biophysik
Charitꢁ-Universitꢂtsmedizin Berlin, Charitꢁplatz 1
10117 Berlin (Germany)
Ga subunit, GtaACHTUNTRGNEUNG(340-350) (Figure 1), binds into a central
crevice in the cytoplasmic domain opened in the active re-
ceptor.[7] A second binding site on the G protein is the pre-
nylated C terminus of the Gg subunit. In the case of Gt, the
Fax : (+49)30-450-524952
E-mail: oliver.ernst@charite.de
binding site Gtg
G
ACHTUNGTRENNUNG(60-
[b] Dr. Y. Chen, Dr. Y. Wu, Prof. Dr. K. Nakanishi
Department of Chemistry, Columbia University
New York, NY 10027 (USA)
71)far).[8] The binding site of Gtg
AHCTUNGTRENNUNG
less clear, although there is multiple evidence that it is in-
volved in initial docking to the receptor.[9]
Fax : (+1)212-932-8273
E-mail: kn5@columbia.edu
Herein, we set out to determine the binding site of Gtg-
[c] Dr. P. Henklein
AHCTUNGTREG(NNUN 60-71)far on light-activated rhodopsin by preparing a syn-
thetic photoactivable peptide derived from the farnesylated
Gtg C terminus. For this purpose, a derivative of the native
GtgACTHNUGRTENUNG(60-71)far peptide (Scheme 1A, peptide 1) was synthe-
sized, which contained a p-ethynylbenzoyl moiety modified
Phe-64, yielding the photoactivable ethynylated benzophe-
none probe (Scheme 1B, peptide 2). The MALDI-TOF-MS
Institut fꢀr Biochemie, Charitꢁ-Universitꢂtsmedizin Berlin
Charitꢁplatz 1, 10117 Berlin (Germany)
[d] Dr. X. Li
Department of Chemical Engineering, Columbia University
New York, NY 10027 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000441.
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with
O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HBTU) activation chemistry and
Fmoc-protected 4-ethynylated p-benzoyl-l-phenylalanine
(Ebp), which was prepared as described in Scheme 2. The
corresponding p-benzoyl-l-phenylalanine is known as a
useful photoactivable probe.[14] The C-terminal cysteine in
peptide 2 was farnesylated by using farnesyl bromide.[15] For
photo-cross-linking and subsequent identification of the in-
teraction site (Scheme 3), the respective photolabile pep-
tide 2 was incubated with native rod cell disc membranes
(rhodopsin represents 90% of the total protein in these
membranes). Orange light was used to form activated rho-
dopsin, and its complex with peptide 2 was cross-linked by
irradiation with 350 nm light. After solubilization in lauryl-
dimethylamine oxide and reduction with dithiothreitol, the
cross-linked protein–peptide complex was alkylated with io-
doacetamide and precipitated with trichloroacetic acid
(TCA). CNBr cleavage (that was used earlier to specifically
cleave rhodopsin at methionine
Figure 1. Crystal structure of the heterotrimeric G protein transducin,
Gtabg (PDB accession 1GOT). Rhodopsin interacting sites are the C-ter-
minal regions of Gtg and Gta (shown in purple and yellow, respectively),
and modeled in their active receptor interacting conformations known
from NMR spectroscopy.[10]
sites)[3d,16] then yielded a mix-
ture of peptide fragments which
were subjected to “click-
chemistry” in the presence of
biotin propyl azide (Schemes 2
and 3). Click chemistry, that is,
CuI-catalyzed chemo-selective
coupling
between
organic
azides and terminal alkynes
(like the ethynyl group of Ebp),
was used to add a biotin-tag to
the cross-linked peptidic frag-
ments for further purification
by biotin-avidin affinity chro-
matography (Scheme 3). The
[3+2] cycloaddition reaction
with the azido group in biotin
propyl azide represents a fast
and selective chemical transfor-
mation under mild conditions.
Only the biotin-tag-derivat-
ized photo-cross-linked rhodop-
sin peptidic fragments bind to
avidin–agarose. These frag-
ments were eluted with 8m gua-
nidine-HCl, desalted, and frac-
tionated on an Atlantis dC18
Scheme 1. Peptide 1: native Gtg
ACHTUNGTREN(NUGN 60-71)far peptide derived from the transducin Gtg subunit
(60DKNPFKELKGGC71-farnesyl). Peptide 2: photoactivable Phe-64 p-ethynylbenzoyl-modified peptide 1.
spectrum of peptide 2 is shown in Figure S1 in the Support-
ing Information.
Position 64 was chosen because earlier studies suggested
that Phe-64 is part of the rhodopsin interacting surface.[11]
Further, a benzophenone group at this position had little
(3 mm, 4.6ꢄ150 mm, Waters) reverse-phase column by
HPLC (Figure S2 in the Supporting Information). Purified
peptides were mixed with a-cyano-4-hydroxycinnamic acid
as the matrix and subjected to MALDI-TOF mass spectrom-
etry. Results of the analysis of the biotin-tag-derivatized
fragments are shown in Figure 2. Three signals correspond-
ing to m/z of 3370.1 (peak 1), 3441.6 (peak 2), and 3485.0
(peak 3) were observed. The rhodopsin peptidic fragment in
peak 1 had an actual m/z value of 1375.7, calculated by sub-
tracting the m/z value of 1994.4 for peptide 2 modified with
biotin propyl azide. A m/z value of 1375.7 (or 1375.0 as de-
effect on the affinity of the GtgACTHNUGRTNEUNG(60-71)far-derived peptide
for active rhodopsin.[12] Introduction of the alkyne group to
the benzophenone allows click chemistry, which has proven
to be useful in labeling biomolecules for in vitro and in vivo
studies, to be performed.[13] Peptide 2 was synthesized by
using the N-(9-fluorenyl)methoxycarbonyl (Fmoc) strategy
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Photo-Cross-Linking Strategy
COMMUNICATION
Scheme 2. Synthesis of the “clickable” amino acid and the corresponding biotinylated conjugate (see the Supporting Information). Fmoc-OSu=N-(9-flu-
orenylmethoxycarbonyloxy)succinimid, TFA=trifluoroacetic acid, DMAP=4-(dimethylamino)pyridine, EDC=N-(3-dimethylaminopropyl)-N’-ethylcar-
bodiimide.
termined in another experiment; Figure S3 in the Support-
ing Information) is consistent with the expected mass of
1374.7 [M+H]+ of a peptide fragment corresponding to the
sequence 144SNFRFGENHAIM155 of rhodopsin.[16] This se-
quence covers the cytoplasmic end of transmembrane
helix 4 and the C-terminal half of the second cytoplasmic
loop connecting transmembrane helices 3 and 4 (Figure 3).
The identification of the other two peaks, probably resulting
from incomplete CNBr cleavage,[17] was not possible, so far,
and in this specific case was hampered by the use of a
highly hydrophobic peptide probe.
Our work aimed at testing a new photo-cross-linking
strategy to map the interaction sites between activated rho-
dopsin and GtgACHTUNGTRENNUNG(60-71)far. Our study extends previous cova-
lent (photo)-cross-linking approaches on rhodopsin/Gt inter-
action using different strategies, that is, reactive groups and
analysis procedure.[3a–c] In previous studies, photoactive re-
agents were attached to the sulfhydryl group of cytoplasmic
monocysteine rhodopsin mutants to primarily map the rho-
dopsin contact sites on the Gta subunit, whereas we have
MALDI-TOF mass spectrometry analysis, resulted in the
identification of the interacting peptide sequence Ser144-
Met155 on rhodopsin, which contains the cytoplasmic end of
helix 4 and half of the second cytoplasmic loop. This loop is
likely to play a role in the two-step interaction between acti-
vated rhodopsin and Gt and is assumed to rearrange accord-
ing to a current modeling study.[9b,18]
In summary, we developed a new photo-cross-linking
strategy that makes use of p-ethynylbenzoyl-modified phe-
nylalanine and exploits click chemistry to purify the photo-
cross-linked products for subsequent mass spectrometric
identification of proteolytic peptide fragments. Expression
systems were developed to generate proteins with genetical-
ly encoded benzophenone by using p-benzoyl-l-phenylala-
nine and amber codon suppression methodology.[19] We envi-
sion that the ethynylated p-benzoyl-l-phenylalanine (com-
pound 9 in Scheme 2) might be incorporated into proteins in
vivo, which adds a high potential to the presented photo-
cross-linking strategy for proteome research.
utilized the benzophenone moiety in Ebp/Phe-64 of GtgACTHNUTRGNEUNG(60-
71)far (Scheme 3). The combination of click chemistry in
the presence of biotin propyl azide as a probe, followed by
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O. P. Ernst, K. Nakanishi et al.
Scheme 3. Strategy for photo-cross-linking of peptide 2 to light-activated rhodopsin (R*) and subsequent analysis of cross-linked sites on rhodopsin. Pep-
tide 2, GtgACHTUNGTRENNUNG(60-71)far peptide with p-ethynylbenzoyl modified Phe-64. Reagent I, biotin propyl azide was prepared by a two-step synthesis (see the Sup-
porting Information).
Experimental Section
General experimental procedure: Bovine retinal rod outer segment mem-
branes were prepared under dim red light[20] and washed with 5m urea to
strip off surface-bound proteins.[21] Washed rod outer segment mem-
branes for extra MII measurements were prepared according to referen-
ce [9b]. THF and diethyl ether were distilled from sodium/benzophenone
ketyl. Dichloromethane was distilled over CaH2. All other reagents and
starting materials were purchased and used as received (Aldrich, St.
Louis, MO). Avidin agarose resin was purchased from ThermoFisher Sci-
entific. Reactions were monitored by analytical TLC using silica gel 60
F254 plates and spots were visualized by UV light irradiation (254 nm)
or oxidative stain (PMA=phosphomolybdic acid). Flash column chroma-
tography was performed by using silica gel (230–400 mesh). Analytical
HPLC was performed on a Waters instrument with UV detector (200–
700 nm) by using a C18 reverse-phase column (Waters, 4.6 mmꢀ150 mm)
to analyze synthetic Gtg-derived peptides. Water and acetonitrile were
used as eluents in various gradient programs at a flow of 0.6 mLminꢀ1
.
1H NMR spectra were recorded with Bruker (300 or 400 MHz) spectrom-
eters. The chemical shifts are expressed in ppm (d) downfield from tetra-
methylsilane (in CDCl3). UV/Vis spectra were recorded at 258C in a
Perkin–Elmer Lambda 40 spectrophotometer. Mass spectra were mea-
sured on a JEOL KMS-HX110/100A HF mass spectrometer under FAB
conditions or a Voyager DE MALDI-TOF mass spectrometer (Applied
Biosystems). Synthetic compounds 3–9, and 13 (see Scheme 2 and de-
scriptions in the Supporting Information) were subjected to FABMS.
Peptide synthesis: This was performed on an ABI 433A automated pep-
tide synthesizer (Applied Biosystems) on a 0.1 mm scale with 300 mg
TentaGel S-RAM-Fmoc-resin (capacity 0.22 mmolg;ꢀ1 RAPP Polymere
GmbH Tꢀbingen, Germany) by using the Fmoc (N-(9-fluorenyl)methox-
ycarbonyl)/tert-butyl alcohol strategy. The following side-chain protecting
groups were used: tert-butoxycarbonyl (Lys), tert-butyl ester (Asp and
Glu) and trityl (Asn and Cys). Couplings were performed with O-(7-aza-
Figure 2. MALDI-TOF mass spectrum of rhodopsin fragments photo-
cross-linked to peptide 2. The photo-cross-linked rhodopsin was digested
with CNBr, labeled with biotinylated reagent I (Scheme 3), purified by
avidin–agarose, and subjected to MS. Inset: the amino acid sequence and
expected mass of the identified rhodopsin fragment (peak 1).
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5 m, 300 ꢅ) with a linear gradient of 10 to 100% B over a period of
45 min (A: water (1000 mL), TFA (2 mL); B: acetonitrile (500 mL),
water (100 mL), TFA (1 mL)) to give the final pure product. Peptide far-
nesylation was carried out as follows: 5 mm peptide was dissolved in 50%
n-propyl alcohol and Na2CO3 was added to a final concentration of
20 mm. The solution was then treated with 30 mm farnesyl bromide in a
10% solution of n-propyl alcohol for 18–24 h at room temperature in the
dark. Farnesylated peptides were purified by reverse-phase chromatogra-
phy by using a PepRPC fast protein liquid chromatography column (GE
Healthcare) using a linear (0–100%) gradient of acetonitrile in water
containing 0.1% TFA. The molecular weight of purified peptides was de-
termined by electrospray mass spectrometry by using a Vestec VT 201
mass spectrometer. Purified peptides were lyophilized and stored at
ꢀ208C under nitrogen. Immediately before the experiments, the peptides
were dissolved in appropriate buffers or deionized water and adjusted to
pH 7 to obtain stock solutions of 1–10 mm.
Modified amino acid synthesis: The modified amino acid was synthesized
by slight modification of a previous route.[12] For the synthesis of p-(4-hy-
droxybenzoyl)phenylalanine, p-bromobenzyl bromide was treated with
(R)-Schollkopf reagent, and then incubated with 4-[(trimethylsilyl)ethy-
nyl]benzaldehyde. Subsequent oxidation, deprotections, and hydrolysis
gave a single diastereomer (100% de) as indicated. Biotin-tag-derivatized
amino acid was obtained by treating biotin propyl azide with above com-
pound.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft SFB
449 (to O.P.E.) and NIH grants GM 34509 (to K.N.).
Keywords: binding sites · G-protein-coupled receptors ·
mass spectrometry
interactions
· photochemistry · protein–protein
Figure 3. Crystal structures (A) of rhodopsin (left, PDB accession
number 1GZM) and opsin (right, PDB accession number 3CAP), and
transmembrane topological model (B) of rhodopsin. Rhodopsin and
opsin contain seven transmembrane helices (blue to orange in (A) and
TM1 to TM7 in (B)), followed by cytoplasmic helix 8 (red in (A) and H8
in (B)), which is terminated by palmitoylated Cys322 and Cys323 (in
opsin only palmitoylation of Cys322 is shown). Oligosaccharides are at-
tached to Asn2 and Asn15 in the N-terminal region. The transmembrane
helices are linked by cytoplasmic (top) and extracellular (bottom) loops.
In (A), C-terminal residues 325–348 are not shown. The rhodopsin/opsin
peptide fragment (144SNFRFGENHAIM155), shown in black (A) and as
filled circles (B), was identified by cross-linking experiments with pep-
[1] A. Sinz, Mass Spectrom. Rev. 2006, 25, 663–682.
[2] K. P. Hofmann, P. Scheerer, P. W. Hildebrand, H. W. Choe, J. H.
Park, M. Heck, O. P. Ernst, Trends Biochem. Sci. 2009, 34, 540–552.
[3] a) J. F. Resek, D. Farrens, H. G. Khorana, Proc. Natl. Acad. Sci.
USA 1994, 91, 7643–7647; b) K. Cai, Y. Itoh, H. G. Khorana, Proc.
Natl. Acad. Sci. USA 2001, 98, 4877–4882; c) Y. Itoh, K. Cai, H. G.
Khorana, Proc. Natl. Acad. Sci. USA 2001, 98, 4883–4887; d) B.
Borhan, M. L. Souto, H. Imai, Y. Shichida, K. Nakanishi, Science
2000, 288, 2209–2212.
[4] a) H. Schwalbe, G. Wess, ChemBioChem 2002, 3, 915–919; b) K. L.
Pierce, R. T. Premont, R. J. Lefkowitz, Nat. Rev. Mol. Cell Biol.
2002, 3, 639–650.
tide 2 (GtgACHTUNGTRENNUNG(60-71)far peptide containing p-ethynylbenzoyl-modified phe-
nylalanine. The identified sequence (Figure 2, inset) covers most of the
second cytoplasmic loop (connecting transmembrane helices 3 and 4) and
the cytoplasmic end of transmembrane helix 4.
[5] K. Palczewski, Annu. Rev. Biochem. 2006, 75, 743–767.
[6] a) K. Palczewski, T. Kumasaka, T. Hori, C. A. Behnke, H. Motoshi-
ma, B. A. Fox, I. Le Trong, D. C. Teller, T. Okada, R. E. Stenkamp,
M. Yamamoto, M. Miyano, Science 2000, 289, 739–745; b) T.
Okada, M. Sugihara, A. N. Bondar, M. Elstner, P. Entel, V. Buss, J.
Mol. Biol. 2004, 342, 571–583; c) J. Li, P. C. Edwards, M. Burgham-
mer, C. Villa, G. F. Schertler, J. Mol. Biol. 2004, 343, 1409–1438;
d) J. H. Park, P. Scheerer, K. P. Hofmann, H.-W. Choe, O. P. Ernst,
Nature 2008, 454, 183–187.
[7] P. Scheerer, J. H. Park, P. W. Hildebrand, Y. J. Kim, N. Krauss, H. W.
Choe, K. P. Hofmann, O. P. Ernst, Nature 2008, 455, 497–502.
[8] O. G. Kisselev, M. V. Ermolaeva, N. Gautam, J. Biol. Chem. 1994,
269, 21399–21402.
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HATU) in N-methylpyrrolidone as a coupling agent. Deprotection of
the Fmoc group was performed during the complete synthesis with 20%
piperidine in DMF. The final deprotection from the resin was performed
with 95% TFA in water containing 3% triisopropylsilane and 5%
phenol. The crude protein was farnesylated with farnesyl bromide and
purified by reverse-phase (RP) HPLC on a 7 m Zorbax SB C18 column
(21.2ꢄ250 mm, Prep HT) with a linear gradient of 50–60% B over
45 min (A: water (1000 mL), TFA (2 mL); B: acetonitrile (500 mL),
water (100 mL), TFA (1 mL)) at a flow rate of 10 mLminꢀ1 with spectro-
photometric monitoring at l=220 nm. The fractions were checked by
RP-HPLC (Shimadzu LC10) on a VYDAC C18 column (4.6ꢀ250 mm,
[9] a) O. G. Kisselev, C. K. Meyer, M. Heck, O. P. Ernst, K. P. Hofmann,
Proc. Natl. Acad. Sci. USA 1999, 96, 4898–4903; b) R. Herrmann,
M. Heck, P. Henklein, P. Henklein, C. Kleuss, K. P. Hofmann, O. P.
Ernst, J. Biol. Chem. 2004, 279, 24283–24290.
Chem. Eur. J. 2010, 16, 7389 – 7394
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7393

O. P. Ernst, K. Nakanishi et al.
[10] a) O. G. Kisselev, M. A. Downs, Structure 2003, 11, 367–373;
b) O. G. Kisselev, J. Kao, J. W. Ponder, Y. C. Fann, N. Gautam, G. R.
Marshall, Proc. Natl. Acad. Sci. USA 1998, 95, 4270–4275; c) B. W.
Koenig, G. Kontaxis, D. C. Mitchell, J. M. Louis, B. J. Litman, A.
Bax, J. Mol. Biol. 2002, 322, 441–461.
[11] O. G. Kisselev, M. A. Downs, Biochemistry 2006, 45, 9386–9392.
[12] Y. Chen, R. Herrmann, N. Fishkin, P. Henklein, K. Nakanishi, O. P.
Ernst, Photochem. Photobiol. 2008, 84, 831–838.
[13] a) M. D. Best, Biochemistry 2009, 48, 6571–6584; b) J. E. Moses,
A. D. Moorhouse, Chem. Soc Rev. 2007, 36, 1249–1262; c) H. Lin,
C. T. Walsh, J. Am. Chem. Soc. 2004, 126, 13998–14003; d) T. S. Seo,
Z. Li, H. Ruparel, J. Ju, J. Org. Chem. 2003, 68, 609–612; e) A. E.
Speers, B. F. Cravatt, Chem. Biol. 2004, 11, 535–546; f) Q. Wang,
T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J.
Am. Chem. Soc. 2003, 125, 3192–3193.
[16] Z. Ablonczy, R. K. Crouch, D. R. Knapp, J. Chromatogr. B 2005,
825, 169–175.
[17] Z. Ablonczy, M. Kono, D. R. Knapp, R. K. Crouch, Vision Res. 2006,
46, 4493–4501.
[18] P. Scheerer, M. Heck, A. Goede, J. H. Park, H. W. Choe, O. P. Ernst,
K. P. Hofmann, P. W. Hildebrand, Proc. Natl. Acad. Sci. USA 2009,
106, 10660–10665.
[19] a) J. W. Chin, A. B. Martin, D. S. King, L. Wang, P. G. Schultz, Proc.
Natl. Acad. Sci. USA 2002, 99, 11020–11024; b) I. S. Farrell, R. Tor-
oney, J. L. Hazen, R. A. Mehl, J. W. Chin, Nat. Methods 2005, 2,
377–384; c) S. Ye, C. Kohrer, T. Huber, M. Kazmi, P. Sachdev, E. C.
Yan, A. Bhagat, U. L. Rajbhandary, T. P. Sakmar, J. Biol. Chem.
2008, 283, 1525–1533.
[20] N. Fishkin, Y. P. Jang, Y. Itagaki, J. R. Sparrow, K. Nakanishi, Org.
Biomol. Chem. 2003, 1, 1101–1105.
[14] a) J. C. Kauer, S. Erickson-Viitanen, H. R. Wolfe, Jr., W. F. DeGra-
do, J. Biol. Chem. 1986, 261, 10695–10700; b) G. Dorman, G. D.
Prestwich, Biochemistry 1994, 33, 5661–5673; c) P. J. Weber, A. G.
Beck-Sickinger, J. Pept. Res. 1997, 49, 375–383.
[21] H. Shichi, R. L. Somers, J. Biol. Chem. 1978, 253, 7040–7046.
[15] O. P. Ernst, C. K. Meyer, E. P. Marin, P. Henklein, W. Y. Fu, T. P.
Sakmar, K. P. Hofmann, J. Biol. Chem. 2000, 275, 1937–1943.
Received: February 19, 2010
Published online: May 20, 2010
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Chem. Eur. J. 2010, 16, 7389 – 7394