Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: Improved labeling and structural implications

Article abstract of DOI:10.1016/j.bmc.2011.10.027

Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a p

Full text of DOI:10.1016/j.bmc.2011.10.027

Bioorganic & Medicinal Chemistry 19 (2011) 7559–7569
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Bioorganic & Medicinal Chemistry
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Photoaffinity labeling of Ras converting enzyme using peptide substrates
that incorporate benzoylphenylalanine (Bpa) residues: Improved labeling
and structural implications
Kelly Kyro a,b, Surya P. Manandhar c, Daniel Mullen a,b, Walter K. Schmidt c, Mark D. Distefano a,b,
⇑
a Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
b Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
c Department of Biochemistry and Molecular Biology, University Georgia, Athens, GA 30602, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing
CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic
signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its
mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide ana-
logues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photo-
affinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near
Received 23 August 2011
Revised 4 October 2011
Accepted 10 October 2011
Available online 18 October 2011
Keywords:
the Rce1p active site. By incorporating the photoactive group via p-benzoyl-L-phenylalanine (Bpa) residues
Photoaffinity labeling
Benzophenone
Bpa
directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a
previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted
photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that resi-
dues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor
design.
Rce1
Prenyl protease
Protein prenylation
CAAX processing
Farnesyl
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
target for inhibition of aberrant cell signaling because it is required
for full biological activity of CAAX proteins in some cases.8 Indeed,
1.1. The CAAX prenyl protease, Ras converting enzyme 1 (Rce1p)
Rce1p modulates Ras membrane localization and inhibition of
Rce1p alters Ras function.9
Ras converting enzyme 1 (Rce1p) modifies CAAX proteins (Ras,
RhoB), essential parts of all mammalian cells. If these proteins be-
come constitutively activated, they can play a role in cancerous sig-
nal transduction.1,2 The first step in CAAX (C = cysteine, A = an
aliphatic amino acid, X = one of several amino acids) protein pro-
cessing is isoprenylation, a cytosolic event. Prenyltransferase en-
zymes transfer either a C15 or C20 (farnesyl or geranylgeranyl,
respectively) isoprenoid lipid to the CAAX cysteine, rendering it
more hydrophobic and often enhancing protein association with
membranes.3,4 This is followed by endoproteolytic trimming of the
–AAX tripeptide by the isoprenyl protein specific protease, Rce1p,
or the functionally related Ste24p.5,6 The final processing step in-
volves carboxylmethylation of the C-terminal prenyl cysteine by
the isoprenylcysteine carboxymethyltransferase (ICMT), also called
Ste14p in yeast.7 Disruption of CAAX protein function has been a
therapeutic target in drug discovery, with most efforts focused on
farnesyltranferase inhibitors (FTIs). Rce1p represents an additional
Rce1p is reported to be an integral membrane protein localized
within the endoplasmic reticulum (ER), and the processing of its
CAAX substrates is an ER membrane-associated event.6,10–12 The
critical catalytic residues of Rce1p are purported to be embedded
in the membrane to facilitate interaction with isoprenoid-modified
substrates, but the structure and topology of Rce1p have yet to be re-
solved.13 Based on sequence alignment studies, Rce1p is predicted to
be heptahelical, possessing seven transmembrane spanning seg-
ments.14 More detailed structural analysis including crystallization
of Rce1p has not been accomplished because of its membrane-
bound nature.12 Despite the fact that integral membrane proteins
constitute 25–30% of the average proteome and approximately
50% of all medically relevant drug targets in humans, they continue
to remain poorly characterized.15 The lack of structural data is due to
difficulties in expression, purification and solubilization as well as
low abundance of protein.15,16 Those problems are compounded
by the challenges associated with crystallization or NMR analysis
of such proteins.
Mutation, inhibition, and bioinformatic studies have been per-
formed to determine the mechanism and protease class of Rce1p.
⇑
Corresponding author. Tel.: +1 612 624 0544; fax: +1 612 626 7541.
E-mail address: diste001@umn.edu (M.D. Distefano).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.027

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K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
Sequential analysis of Rce1p identified certain invariant residues
(i.e., E156, H194, and H248 of yeast Rce1p) that when mutated, were
found to be necessary for normal Rce1p activity.13,14,17 Analogous
glutamate and histidine residues are necessary for Trypanosoma
brucei Rce1p activity, highlighting their significance for proper
Rce1p function.11 These residues are also conserved among a family
of related prokaryotic proteins that largely have unknown func-
tion.18 The confirmation that yeast Rce1p was inhibited by 1,10-
phenanthroline and excess zinc further supports the possibility of
a metal-dependent protease mechanism.12,13 However, until its
structure is fully characterized, the proteolytic mechanism of Rce1p
remains uncertain.12
cleavage of the –K(Dnp)IM tripeptide, and the time-dependent
increase in fluorescence is used to monitor the rate of
endoproteolysis.
Compounds 4 (previously reported by our lab) and 6 (Fig. 2) are
analogues of 2, containing photoactive benzophenone-modified
isoprenoids linked to the peptide; in each case, the benzophenone
group is tethered to the isoprenoidal portion of the probe by a sta-
ble ether bond to permit photocrosslinking of Rce1p.27,33,34
Peptide 1 (Fig. 2), containing a free cysteine thiol, was synthe-
sized as previously described and alkylated with farnesyl bromide
to create peptide 2 (Fig. 2).35,36 The aryl ketone-containing peptide
photophores, 4 and 6 (Fig. 2), were prepared in a similar fashion
using benzophenone-modified isoprenoids 3 and 5 (Fig. 2).37,38
Biotin was incorporated on the probes to allow for selective
purification of cross-linked protein via streptavidin pull-down.
Peptide 7 (Fig. 3) containing an N-terminal biotin linked to the
peptide via a PEG3 spacer and a Dde-protected lysine (to allow
for introduction of a fluorophore) was synthesized as previously
described,35 followed by alkylation with 3 and 5 (Fig. 2) to obtain
peptides 8 and 9.
Probes 11–14 (Fig. 4) were developed in order to explore the
efficiency of labeling when the photoactive benzophenone group
is incorporated into the actual peptide substrate sequence. In these
molecules, the native, farnesylated K-Ras4b sequence was retained
in all respects except for substitution of the non-natural, photore-
active amino acid, p-benzoyl-L-phenylalanine (Bpa), at specific
(P6–P3) positions upstream of the scissile bond on the peptide. A
biotin affinity tag was again introduced at the N-terminus.
1.2. Selective labeling of Rce1p using affinity-based
photoprobes
Multiple approaches have been used in attempts to characterize
the structure and mechanism of Rce1p. Both competitive inhibitors
and active-site directed, irreversible affinity-labeling reagents have
been designed, but these have not yet yielded a consistent picture for
a mechanism.19–21 Site-directed mutagenesis has also been useful in
identifying amino acids that are necessary for Rce1p function.13,14
However, mutation of residues in the enzyme sequence can cause
architectural changes that affect enzyme–protein interactions or
otherwise compromise the natural processing activity of an enzyme.
An alternative approach for characterizing protein–ligand binding is
the utilization of photoactivatable analogues that mimic the natural
substrate protein. This technique has been used successfully in pro-
filing several systems, including G protein coupled receptors
(GPCRs) and metalloproteases.22–25 Lipid-modifying enzymes can
also be labeled without compromising activity using affinity-based
probes that incorporate a photoreactive group capable of crosslink-
ing to nearby residues upon irradiation.16,26 The irreversible cova-
lent linkage formed between the photoprobe and target protein
allows for structural characterization of the resulting enzyme–
probe interaction. Enzymatic digestion ofthe covalentphotoadducts
formed in the cross-linking reaction followed by LC–MS–MS
sequencing enables identification of labeled peptide fragments.27,28
Benzophenone photophores have been used broadly in photo-
affinity-labeling applications because of their chemical stability
and wavelength of activation (365 nm), which is not damaging to
proteins.27,29,30 In particular, benzophenone-containing Bpa (p-ben-
2.2. HPLC and MS–MS analysis of enzymatic processing of 6
Peptide 6 was evaluated as an Rce1p substrate and the cleavage
activity was characterized using RP-HPLC. Peptide 6 (40
lM) was
incubated with Rce1p-containing membranes (25 g/mL) at 35 °C
l
for approximately 4 h in a large-scale (1–2 mL) proteolytic assay
that generated the Rce1p-catalyzed proteolysis product 10 (Fig. 5,
left). Use of an HPLC instrument equipped with a diode array detec-
tor allowed for simultaneous evaluation of two UV wavelengths. The
220 nm wavelength (Fig. 5, right, solid trace) permitted general
visualization of peptide and the 349 nm wavelength (Fig. 5, right,
dotted trace) revealed specific information on the presence or ab-
sence of the Dnp fluorophore. Peaks at tR = 46.5 min in both
220 nm and 349 nm traces indicate unprocessed peptide 6 with an
intact Dnp-containing CAAX sequence. Loss of the Dnp group at
tR = 28 min in product 10 is denoted by loss of absorbance at
349 nm (Fig. 5, right, dotted trace) indicating Rce1p-catalyzed cleav-
age of the –K(Dnp)IM tripeptide. However, a peak at tR = 28 min is
still visible at the 220 nm wavelength (Fig. 5, right, solid trace) sug-
gesting the formation of product 10 after loss of the –AAX tripeptide.
Purified unprocessed peptide 6 was characterized by ESI-
MS–MS (see Fig. 1A of Supplementary data). The most abundant,
triply-charged [M+3H]3+ parent ion (m/z = 566.6) in the ESI-MS
was selected for MS–MS sequencing of 6. Fragmentation of that
ion yielded several b-, and y-type fragments; eight out of nine
b-type ions and five out of nine y-type ions were observed confirm-
ing the proposed structure of 6 (see Table 1 in the Supplementary
data for complete listing and assignments). The most abundant
ions in the ESI-MS–MS of 6 were doubly-charged parent ions that
had lost benzophenone (-d) (m/z = 743.4) and C10-prenyl benzo-
phenone (-e) (m/z = 676.3) (Supplementary data, Fig. 1A). Cleavage
of the thioether bond linking the benzophenone isoprenoid to the
peptide (-e) was the most common mode of fragmentation. In
addition, very intense ions from benzophenone fragments (BP)
(m/z = 195.1) and (d) (m/z = 213.1) were observed.
zoyl-L-phenylalanine) residues have proven useful because of their
ease of synthesis and high crosslinking efficiency.24,25,27 In earlier
work, we reported on the use of a compound that incorporated a
benzophenone-functionalized isoprenoid into a peptide substrate
for Rce1p. In this study, we have extended our earlier work by pre-
paring a similar probe employing a longer isoprenoid spacer. We
have also explored the utility of benzophenone-functionalized pep-
tides by directly substituting Bpa residues into the native, farnesy-
lated K-Ras4b sequence (Fig. 1). This latter design greatly
increased the efficiency of Rce1p labeling relative to our earlier
work.
2. Results and discussion
2.1. Design of Rce1p substrate analogues incorporating
benzophenone photophores
The internally quenched fluorogenic peptide substrate 2 (Fig. 2)
is a farnesylated nona-peptide based on the K-Ras4b C-terminal se-
quence, KSKTKC(farnesyl)VIM, that has been used in proteolysis
assays to monitor Rce1p activity.10,12,31,32 It contains an N-terminal
Abz (2-aminobenzoyl) fluorophore and a dinitrophenyl quencher
(e
-dinitrophenyl lysine) that replaces the P10 (V) position.31
The Rce1p-catalyzed proteolysis product, 10, was also charac-
terized by ESI-MS–MS. A large-scale assay with 6 was performed
The peptide has higher fluorescent yield upon Rce1p-catalyzed

K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
7561
NH2
NH2
NH2
S
O
O
O
O
Streptavidin
Biotin-PEG3
H
N
H
N
H
N
H
N
OH
N
H
N
H
NH
N
H
N
H
O
O
O
O
O
OH
S
O
Photoactivatable crosslinker
1) UV light
2) Streptavidin Pull-down (SPD)
3) Western Blot visualizing HA-tag on Rce1p
Figure 1. Photoactivatable peptide substrate for photoaffinity labeling of Rce1p containing a biotin-PEG3 spacer for SPD isolation of cross-linked protein.
NO2
HN
NO2
NH2
NH2
NH2
S
NH2
O
O
O
O
O
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
N
H
H
O
O
O
O
O
OH
OH
SR
1: R = H
2: R = Fr =
O
O
Br
Br
3
5
4: R = C5BP =
6: R = C10BP =
O
O
O
O
O
O
Figure 2. Structures of isoprenylated peptide substrate, 2, derived from the C-terminal sequence of mammalian K-Ras4b, benzophenone-modified isoprenoids 3 and 5 used
for peptide functionalization, and substrate analogues 4 and 6, incorporating benzophenone-modified isoprenoids.
NO2
HN
NO2
NH2
NH2
NH2
S
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
N
N
H
Biotin-(PEG)3
H
O
O
O
O
O
O
OH
OH
SR
7: R = H
HN
O
O
O
8: R = C5BP =
9: R = C10BP =
O
O
O
Figure 3. Structures of biotinylated peptide 7, containing a free thiol, and 8 and 9, following alkylation using the benzophenone-modified isoprenoids, 3 and 5.
as above in order to generate 10 and evaluate its MS fragmenta-
tion. The assay material was purified using reversed phase chroma-
tography and analyzed by ESI-MS. Direct infusion of the products
obtained from Rce1p-catalyzed proteolysis of 6 revealed that the
dominant [M+2H]2+ species (m/z = 580.4) in the ESI-MS was
consistent with the expected product 10 (Fig. 5, left). MS–MS

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K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
NH2
NH2
O
S
O
O
O
O
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
Biotin-PEG3
O
O
O
O
O
OH
OH
11
S
NH2
NH2
NH2
S
O
O
O
O
H
N
H
N
H
N
H
N
OH
NH
O
N
N
N
H
N
H
Biotin-PEG3
H
H
O
O
O
O
O
OH
12
S
NH2
NH2
O
S
O
O
O
O
H
N
H
H
N
H
N
N
OH
NH
N
N
H
N
N
Biotin-PEG3
H
H
H
O
O
O
O
O
OH
OH
13
S
NH2
NH2
NH2
S
O
O
O
O
H
N
H
N
H
N
H
N
OH
NH
N
H
N
H
N
N
Biotin-PEG3
H
H
O
O
O
O
O
OH
14
S
O
Figure 4. Rce1p substrate analogues incorporating a photoactivatable Bpa residue at alternating P6 (11)–P3 (14) positions proximal to the naturally recognized
farnesyl-cysteine moiety.
6
1200
1000
800
600
400
200
0
1200
1000
800
600
400
200
0
NH2
NH2
NH2
10
O
O
O
O
S
NH2
H
N
H
N
H
N
N
H
N
H
N
H
OH
O
O
O
OH
OH
10
20
30
40
50
60
O
Time (min)
O
Figure 5. Left: Structure of product 10 derived from Rce1p-catalyzed proteolysis of 6. Right: RP-HPLC chromatogram of Rce1p proteolytic processing of 6. Conversion of 6 to
10 is illustrated by the loss of absorbance at 349 nm corresponding to loss of Dnp upon Rce1p-catalyzed cleavage of the –AAX tripeptide. Solid trace: 220 nm; dotted trace:
349 nm.

K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
7563
fragmentation of that ion resulted in the formation of b- and y-type
2.4. Analysis of labeling efficiency of Rce1p with photoactive
peptides
ions all consistent with the proposed structure for 10 (see Fig. 1B of
Supplementary data). In total, five out of six b-type ions and five
out of six y-type ions were observed (see Table 1 in theSupplemen-
tary data). As was seen in the fragmentation of 6, a large number of
ions where loss of the benzophenone (-b, -d) and prenyl-benzo-
phenone (-c and -e) moieties had occurred were observed, includ-
ing ions related to the doubly-charged parent ion (m/z = 474.4)
(Supplementary data, Fig. 1B). Again, very intense ions from benzo-
phenone fragments (BP) (m/z = 195.1) and (d) (m/z = 213.1) were
also noted. These ions may be useful as signature ions in future
MS analysis for identification of cross-linked species.
Following confirmation that the new benzophenone-modified
isoprenoid 6 could serve as a substrate for Rce1p, the biotinylated
derivatives 8 and 9 (Fig. 3) were evaluated as photoaffinity-
labeling probes. Given that the goal of these experiments was to
increase labeling efficiency, it was also decided to evaluate the
Bpa-containing farnesylated K-Ras peptides 11–14 (Fig. 4) in which
the photoactive group was integrated within the peptide rather
than the isoprenoid. Thus, solutions of 8 and 9 and 11–14 were di-
luted to 15
l
M and irradiated in the presence of Rce1p-enriched
membranes (25
lg/mL) for 20 min at 4 °C. The sample was sub-
2.3. Fluorescence-based kinetic analysis of enzymatic
processing of 6
jected to protein precipitation, which facilitated removal of unin-
corporated probe, followed by streptavidin pull-down designed
to concentrate crosslinked Rce1p species. The recovered protein
was fractionated by SDS–PAGE and analyzed by Western blotting
using antibodies directed against HA, an epitope tag incorporated
into Rce1p via recombinant methods.13
Irradiation of the probes in the presence of Rce1p-containing
membranes resulted in the appearance of bands at approximately
41 kDa, the predicted mass of HA-epitope tagged Rce1p (Fig. 7).
Comparison of the labeling obtained with 9 (Fig. 7, lane 2) with
that of previously described35 probe 8 (Fig. 7, lane 1) revealed that
probe 9 labeled with significantly less efficiency (fourfold less)
than was observed with 8. By contrast, the new Bpa-containing
probes 12 and 13 (see Fig. 7, lanes 4 and 5) labeled Rce1p with effi-
ciencies comparable with probe 8. Excitingly, the labeling efficien-
cies of 11 and 14 (see Fig. 7, lanes 3 and 6) were significantly
improved (up fourfold) compared with 8. Due to its superior solu-
bility, 14 was chosen for use in subsequent labeling experiments.
The aforementioned fluorescence-based proteolysis assay31,32
was previously used to evaluate Rce1p proteolytic processing of
4 and obtain kinetic parameters.35 For purposes of comparison,
the corresponding parameters for 6 were evaluated using the same
method.
Carbonate washed, Rce1p-enriched membranes isolated from
yeast over-expressing yeast Rce1p as the sole CAAX protease10,12
were added to a solution containing the peptide substrates. Rce1p
activity was monitored using a fluorescence microplate reader to
measure the rate of fluorescence production upon cleavage of the
substrate (i.e., release of the -K(Dnp)IM tripeptide). Kinetic evalua-
tion demonstrated that 6 is an Rce1p substrate with a KM of
9.6 lM and a Vmax of 0.85 nmol/min/mg protein (Fig. 6 and Table
1). Comparison of those values with kinetic parameters obtained
using 2 and 4 suggests that the binding affinity of 6 is fourfold less
than that of 4 but compound 6 binds to Rce1p with similar affinity
as 2 (Table 1). The maximal processing rate (Vmax) of 6 is approxi-
mately sevenfold less than that obtained with 2 but ninefold greater
than that of 4. In addition, the efficiency (Vmax/KM) of Rce1p process-
ing of 6 is approximately double that of 4 but 10-fold lower than that
of 2. The kinetic data was fit to the Hill Equation instead of the
Michaelis–Menten Equation since it provided a better fit for the sig-
moidally shaped curves.35 Interestingly, the Hill coefficient, h (Table
1) for 6 was 1.8, somewhat smaller than the values of 2.7 obtained
for 2 and 2.8 for 4, but still greater than unity. The kinetic data sug-
gest that Rce1p is demonstrating positive cooperative binding and
may possess multiple substrate binding sites. Given the impure nat-
ure of the Rce1p used in these experiments, it is not possible to fur-
ther interpret these results on a molecular level in greater detail;
consequently, the significance of these results remains unclear.
2.5. ESI-MS–MS analysis of Bpa-containing peptides 11–14
Probes incorporating Bpa into the peptide backbone (11–14)
versus the isoprenoid moiety (8–9) appeared to yield better overall
photolabeling of Rce1p. Hence, the MS–MS spectra of these com-
pounds were evaluated to facilitate analysis of cleavage products.
Accordingly, peptides 11–14 were sequenced by ESI-MS–MS to
confirm their structures prior to proteolytic cleavage by Rce1p.
The most abundant, doubly-charged [M+2H]2+ parent ions in the
ESI-MS (m/z = 897.7; m/z = 918.3; m/z = 897.6) were selected for
fragmentation of 11, 12, and 13, respectively, by MS–MS. The most
abundant, triply-charged [M+3H]3+ parent ion (m/z = 607.8) was
selected for fragmentation of 14 by MS–MS. Fragmentation of
these ions yielded several b-type and some y-type fragments. Se-
ven out of nine b-type ions were observed in the spectrum of 11.
In the spectra of 12 and 13, a full set of b-type ions (nine out of nine
b-type ions) were observed. The spectrum of 14 revealed eight out
of nine b-type ions and three out of nine y-type ions. Consistently
abundant in all of the spectra were doubly-charged parent ions
that had lost the farnesyl group (m/z = 795.4, 816.1, 795.4, and
808.9; 11–14, respectively). In addition, a very intense ion from a
biotin fragment (B) (m/z = 270.1) was observed in all of the spectra
as well as ions representing loss of the biotin fragment (-B)
(C12H20N3O2S+) and loss of the farnesyl group (-f) (C15H25) (see
Supplementary data for ESI-MS–MS spectra and Table 2 for
complete listing and assignments).
1
0.8
0.6
0.4
0.2
0
2.6. HPLC and MS-MS analysis of enzymatic processing of 14
Because the best labeling results obtained were with probe 14, a
compound containing a non-natural residue within the peptide
sequence, we thought it was important to verify that 14 was in-
0
10
20
6 (µM)
30
40
50
deed a substrate for Rce1p. Accordingly, 14 (20
lM) was incubated
Figure 6. Kinetic analysis of Rce1p-catalyzed proteolysis of benzophenone-mod-
ified substrate 6. Rate versus substrate concentration using peptide 6.
with Rce1p-containing membranes (25 g/mL) at 35 °C for
l

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K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
Table 1
Kinetic parameters observed for Rce1p with benzophenone-modified photoprobes 4 and 6 and farnesylated peptide 2
Substrate
KM
(lM)
Vmax (nmol/min/mg protein)
Vmax/KM (L/min/mg protein)
h
2 (n = 2)a
4 (n = 5)a
6 (n = 5)
6.4 0.2a
1.9 0.3a
9.6 2.0
5.6 0.1a
8.6 ꢀ 10ꢁ4
4.8 ꢀ 10ꢁ5
8.9 ꢀ 10ꢁ5
2.7a
2.8a
1.8
0.093 0.005a
0.85 0.1
a
From Kyro, et al.;35 n = replicate; h = Hill coefficient.
1200
1000
800
600
400
200
0
8
9
11
12
13
14
Figure 7. Western blot analysis (left) and densitometric quantification (right) of photolabeled Rce1p using probes 8, 9 and 11–14, detected with anti-HA following SPD and
SDS–PAGE separation. Lanes 1-6 correspond to Rce1p-containing membranes (RCE1-HA) photolyzed with probes 8, 9 and 11–14 (all at 15
represent densitometric data obtained from three replicate experiments using probes 8, 9 and 11–14.
lM), respectively. Columns
approximately 4 h in a large-scale (1–2 mL) proteolytic assay to
generate the Rce1p-catalyzed proteolysis product 15 (Fig. 8, left)
and analyzed by RP-HPLC. The smaller peak at tR = 64.5 min in
the HPLC trace (Fig. 8, right) indicates unprocessed peptide 14 with
an intact CAAX sequence. The larger peak at tR = 60.5 min with
lower retention time is product 15 (after loss of the -VIM tripep-
tide), confirming that 14 is indeed a bona fide substrate. Impor-
tantly, the fact that 14 is an alternative substrate for Rce1p is
consistent with the hypothesis that this probe is labeling the active
site of the enzyme upon photolysis. A 79% conversion indicating a
4:1 ratio of product to starting material was determined by inte-
grating the full-length and product peaks in the chromatogram.
It should be noted that the extent of conversion of 14 to 15
described here (Fig. 8, right) is greater than that observed for the
processing of 6 to 10 (Fig. 5, right) carried out under similar condi-
tions; this suggests that 14 is a more efficient substrate than 6 for
yeast Rce1p. Since the main objective of the studies reported here
was to identify probes that crosslink with greater efficiency, we did
not pursue more detailed kinetic analyses of these compounds.
To confirm the identity of product 15, it was evaluated by MS
fragmentation by comparison to 14 (see Fig. 2 of Supplementary
data). The assay material was purified using reversed phase
chromatography and analyzed by ESI-MS. The most abundant
[M+2H]2+ species (m/z = 739.4) was selected for MS–MS fragmen-
tation and was consistent with the expected product 15
(Supplementary data, Fig. 2B). A full set of b-type ions (six out of
400
350
15
300
250
200
NH2
NH2
NH2
150
100
50
14
O
O
O
S
H
N
H
N
H
N
NH
N
H
N
H
OH
Biotin-PEG3
O
O
O
15
OH
0
30
40
50
60
70
O
Time (min)
Figure 8. Left: Structure of product 15 derived from Rce1p-catalyzed proteolysis of 14. Right: RP-HPLC chromatogram illustrating a 4:1 conversion (79%) of 14 to 15 after 4 h
of incubation and proteolytic processing by Rce1p.

K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
7565
six b-type ions) were generated along with several y-type ions
(three out of six y-type ions) (see Table 3 of Supplementary data
for complete listing and assignments). As was seen for the spec-
trum of 14 Supplementary data, Fig. 2A), the spectrum of 15 Sup-
detergent concentration used in the pull-down decreased the non-
specific labeling but it could not be completely eliminated.
2.9. Competition experiments using 2 and 14
plementary data, Fig. 2B) contained
a very intense doubly-
charged parent ion that had lost the farnesyl group (m/z = 637.3).
The characteristic biotin fragment (B) (m/z = 270.1) was also seen
along with ions indicating loss of either the biotin fragment (-B)
or the farnesyl (-f).
As further validation of 14 being an active site probe, a compet-
itive experiment was performed using 14 and substrate 2 (Fig. 2), a
known Rce1p substrate that lacks a photolabile Bpa group and con-
tains a farnesyl cysteine. Irradiation of Rce1p under mixed sub-
strate conditions (15
diminution in cross-linked Rce1p relative to 14 alone (84% labeling
observed, Fig. 10). With 100 M of competitor 2, the labeling was
almost completely abolished (7% labeling observed), indicating a
dose-dependent inhibition of labeling. These results suggest that
the two compounds were competing for the same binding site in
Rce1p and that probe 14 is labeling the active site of Rce1p.
lM 14 and 50 lM 2) resulted in a slight
2.7. Photolysis time course to determine maximal labeling of
Rce1p
l
Next, the optimal irradiation time for maximal cross-linking of
Rce1p was determined in a time course study where 14 (15 lM)
was photolyzed with Rce1p-containing membranes for varying
amounts of time (0–60 min) (see Fig. 3 of Supplementary data).
As the photolysis time was increased from 0 to 30 min, there was
a corresponding increase in Rce1p labeling. The most abundant
labeling time appeared to be 30 min Supplementary data Fig. 3,
lane 3), after which the amount of labeling gradually decreased.
Consequently, 30 min was chosen as the optimal labeling time
for Rce1p and was used in subsequent photolysis experiments.
Theoretically, increasing the duration of UV exposure should
result in more abundant labeling. In a study done by Sato and
co-workers, cross-linking experiments were performed for up to
10 h using Bpa-containing protein with no decrease in labeling or
radiation damage.24 However, in our labeling reactions we
observed a decrease in labeling after 30 min of irradiation. We
attribute the decrease in labeling over time to proteolytic degrada-
tion or aggregation of the membrane material during the photoly-
sis step.
Post-photolysis, proteolytic degradation was minimized by
inclusion of protease inhibitors in all subsequent steps of the pro-
cedure, which were also performed on ice. By evaluating the un-
bound fraction from the streptavidin pull-down via Western blot,
it was confirmed that proteolysis had indeed occurred during pho-
tolysis. Following 30 min of irradiation, increasing photolysis time
resulted in a decrease of the 41 kDa Rce1p signal with a concurrent
increase in the abundance of lower molecular weight bands
thought to be proteolytic fragments. The longest photolysis times
yielded general degradation of all of the bands and loss of most
of the HA signal (data not shown).
3. Conclusions
In earlier work, we synthesized and characterized the benzo-
phenone-functionalized isoprenoids 4 and 8 and confirmed their
efficacy as Rce1p substrates. In this study, we synthesized novel,
photolabile Rce1p substrates designed to optimize the photolabel-
ing efficiency of benzophenone-containing probes. By increasing
the length of the isoprenoidal portion of 4 to create 6, the Rce1p
processing efficiency (Vmax/KM) of the benzophenone-modified iso-
prenoids was increased approximately twofold with little impact
on KM (9.6 lM and 6.4 lM for 6 and 2, respectively). However,
photolabeling experiments with the new probe did not result in
improved levels of crosslinking. Hence, we explored the utility of
a series of photoactivatable, Bpa-containing probes that retained
the naturally-recognized farnesyl-cysteine moiety. Evaluation of
the cross-linking efficiency of these probes revealed that all of
the probes labeled Rce1p, with the most intense labeling observed
with probes 11 and 14. Importantly, the amount of cross-linking
obtained with 14 was over fourfold greater than that obtained with
the previously described probe 8. The ability of Rce1p to process 14
to its expected product 15 was confirmed by a combination of RP-
HPLC and ESI-MS–MS analysis. The specificity of 14 for the Rce1p
2.8. Photoaffinity-labeling of Rce1p using Bpa-containing
substrate 14
With the determination of an optimal labeling time of 30 min,
photoaffinity-labeling of Rce1p and two null strains was employed
in order to verify that the protein labeled by probe 14 was indeed
Rce1p. As in the above experiments, a carbonate-washed mem-
brane fraction from Saccharomyces cerevisiae was used as the
source for Rce1p (RCE1-HA in rce1
control experiments were performed using membranes isolated
from strains lacking both CAAX proteases (rce1 ste24 ) or both
CAAX proteases and the yeast ICMT (rce1 ste24 ste14 ).
D ste24D background). Negative
D
D
D
D
D
Specific labeling of Rce1p was observed when 14 was photo-
lyzed in the presence of Rce1p-containing membranes, as evi-
denced by the anti-HA-reactive band observed at 41 kDa (Fig. 9,
lane 6). This band was absent in the control strains lacking Rce1p
either in the absence (Fig. 9, lanes 1 and 3) or presence (Fig. 9, lanes
2 and 4) of irradiation. When Rce1p was treated with 14 in the ab-
sence of irradiation (Fig. 9, lane 5), a small amount of Rce1p was
visualized, attributable to nonspecific binding of Rce1p to the
streptavidin resin employed in the pull-down. Increasing the
Figure 9. Western blot analysis of photolabeled products generated by probe 14 as
detected with anti-HA following SPD and SDS–PAGE separation. Lane 1: Mem-
branes lacking Rce1p (rce1
lacking Rce1p (rce1 ste24
Rce1p, Ste24p, and Ste14p (rce1
Membranes lacking Rce1p, Ste24p, and Ste14p (rce1
D
D
ste24
) with UV irradiation. Lane 3: Membranes lacking
ste24 ste14 ) and no UV irradiation. Lane 4:
ste24 ste14 ) and UV
D) and no UV irradiation. Lane 2: Membranes
D
D
D
D
D
D
D
irradiation. Lane 5: Rce1p-containing membranes (RCE1-HA) and no UV irradiation.
Lane 6: Rce1p-containing membranes (RCE1-HA) and UV irradiation. Compound 14
was used at 15 lM in all instances.

7566
K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
Anaspec. Yeast protease inhibitor cocktail (P8215) and all other re-
(A)
(B)
agents were from Sigma Aldrich. Reactions were performed at room
temperature unless otherwise noted. RP-HPLC analysis was carried
out using a Beckman Model 125/168 instrument equipped with a
diode array UV detector. Preparative RP-HPLC separations employed
a Phenomenex Luna 10
Analytical RP-HPLC separations employed a Varian Microsorb-MV
l
100 Å C18(2) column (10.00 mm ꢀ 25 cm).
5
5
l
l
100 Å C18 column (4.6 mm ꢀ 25 cm) with a Phenomenex Luna
guard cartridge (4.6 mm ꢀ 3 cm). Retention times (tR) were
2
1200
(0 µM)
based on analytical RP-HPLC using a linear gradient from 100%
H2O to 40% H2O/70% CH3CN over 70 min and a flow rate of 1.0 mL/
min. Peptide concentrations were determined by mass (11–14) or
by UV spectroscopy of the Dnp quencher (6 and 9) (349 nm,
2
1000
800
600
400
200
0
(50 µM)
e
= 18,000 cmꢁ1 Mꢁ1, pH 7). The fluorogenic peptides were pro-
tected from light as much as possible to avoid photobleaching of
the fluorophore. C18 Sep-Pak columns were purchased from Waters
and protein precipitation kits were purchased from CalBiochem.
Spin columns, streptavidin resin, and ECL substrate solutions were
purchased from Pierce. Autoradiographic film was purchased from
Bioexpress. Antibodies were purchased from Covance and GE
Healthcare. Densitometry measurements were performed using a
Biorad Molecular Imager, FX phosphorimager.
2
(100 µM)
1
2
3
4.1.1. Yeast strains and plasmids
Figure 10. Photoaffinity labeling and chemiluminescent detection of Rce1p under
competing substrate conditions. (A) Western blot analysis of photolabeled Rce1p
with probe 14 (15 lM) in the absence or presence of competitor 2 (50 or 100 lM) as
detected with anti-HA following SPD and SDS–PAGE separation. Lane 1: Rce1p-
The parent yeast strain used in this study is SM3614 (MATa trp1
leu2 ura3 his4 ste24::LEU2rce1::TRP1).42 SM3614 was transformed
with plasmid pRS316 (CEN URA3) or pWS479 (2
lURA3PPGK-RCE1::-
HA) to create rce1
D
and wildtype (WT) strains, respectively.13,43
containing membranes (RCE1-HA) with UV irradiation. Lane 2: Rce1p-containing
membranes (RCE1-HA) and farnesylated competitor 2 (50
Lane 3: Rce1p-containing membranes (RCE1-HA) and farnesylated competitor 2
(100 M) with UV irradiation. (B) Densitometric quantification of photolabeled
Rce1p. Columns represent densitometric data obtained from four replicate exper-
iments. Sample ordering is the same as in panel A.
lM) with UV irradiation.
Yeast transformations were carried out according to established
methods.44 Transformed yeast strains were grown at 30 °C in syn-
thetic complete medium lacking uracil (SC-Ura).
l
4.1.2. Abbreviations
Abz, 2-aminobenzoyl; BP, benzophenone; Bpa, p-benzoyl-L-
active site was further supported by competition experiments uti-
lizing substrate 2, whose presence reduced photolabeling of Rce1p
in a dose-dependent manner, suggesting that labeling does indeed
occur in the enzyme active site.
phenylalanine, C5BP, 3-methylbenzophenone prenyl ether; C10BP,
3-methylbenzophenone geranyl ether; CLEAR, cross-linked
ethoxylate acrylate resin; Dde, 1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene) ethyl; DI, deionized; DIEA, diisopropylethylamine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Dnp,
dinitrophenyl; ECL, enhanced chemiluminescence; ER, endoplasmic
reticulum; ESI-MS, electrospray ionization mass spectrometry; FA,
formic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; f, farnesyl; GPCR,
G protein coupled receptor; HA, hemagglutinin; HRP, horseradish
peroxidase; BME, 2-mercaptoethanol; OAc, acetate; PBS, phosphate
buffered saline; PEG, polyethylene glycol; PVDF, polyvinylidene
difluoride; PMSF, phenylmethanesulfonyl fluoride; RIPA, radioim-
munoprecipitation assay; RP-HPLC, reversed-phase high pressure li-
quid chromatography; SA, streptavidin; SDS, sodium dodecyl
sulfate; SPD, streptavidin pull-down; SPPS, solid-phase peptide syn-
thesis; TBST, Tris-buffered saline containing Tween 20; TFA, trifluo-
roacetic acid; TFP, tetrafluorophenyl.
Through this study, we have successfully demonstrated in-
creased photolabeling efficiency of benzophenone-containing
Rce1p substrate peptides. By incorporating biotin on the N-terminus
as an affinity tag for purification, retaining the naturally recognized
farnesyl-cysteine, and incorporating a photolabile Bpa residue into
the peptide sequence of an established substrate sequence, the yield
of cross-linked product was significantly increased. This approach
has potential utility for further investigation into the mechanism
and active site identification of Rce1p. By increasingthe labeling effi-
ciency of substrate photoprobes, further structural information on
Rce1p may be obtained via isolation and characterization of the
covalent enzyme–probe complex. This strategy for probe design
may also be useful for studies of other enzymes that act on prenylat-
ed proteins including Ste24p and ICMT.3,39–41 Finally, it is important
to note that the crosslinking results reported here suggest that res-
idues outside the CAAX-box are in contact with Rce1p despite the
fact that the minimal substrate recognized by Rce1p appears to be
an isoprenylated CAAX tetrapeptide.3,14,32 If these upstream interac-
tions involve any specific contacts with the enzyme, it may be fruit-
ful to incorporate such features in future inhibitor designs.
4.2. Chemical synthesis
4.2.1. Synthesis of Abz-KSKTKC(C10BP)K(Dnp)IM, 6
Abz-KSKTKCK(Dnp)IM,35 1 (10.0 mg, 7.4
lmol, 1.0 equiv) was
dissolved in 10.0 mL of 2:1:1 DMF/n-butanol/0.1% aqueous TFA
(v/v/v) and (3-((((2E,6E)-8-bromo-2,6-dimethylocta-2,6-dien-1-
4. Experimental
4.1. General
yl)oxy)methyl)phenyl)(phenyl)methanone, 5 (13.0 mg, 30
4.0 equiv), synthesized according to published methods,33,45 was
added. Zn(OAc)2 (8.0 mg, 37 mol, 5.0 equiv) was dissolved in a
small amount 0.1% aqueous TFA (200 L) and added to the reac-
lmol,
l
l
Analytical TLC was performed on precoated (0.25 mm) Silica Gel
60F-254 plates purchased from E. Merck. Flash chromatography sil-
ica gel (60–120 mesh) was obtained from E. M. Science. TFP-PEG3-
Biotin was from Thermo Scientific. Bpa peptides (11–14) were from
tion. The reaction was monitored by RP-HPLC and was complete
in 2.5 h. The crude material was diluted with 0.1% aqueous TFA
and purified by RP-HPLC using 0.1% TFA/H2O (solvent A) and
0.1% TFA/CH3CN (solvent B) and the following elution profile

K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
7567
performed at 5.0 mL/min: 5.0 min 0% solvent B; linear gradient
from 0% to 60% solvent B over 120 min. The solvent mixture was
removed by lyophilization yielding 1.3 mg (10%) of 6. Purity by
RP-HPLC: 92.6%, tR = 57.5 min, ESI-MS (m/z): [M+2H]2+ calcd for
tR = 46.5 min (51% B), ESI-MS (m/z): [M+2H]2+ (mono) calcd for
C82H122N16O19S2 849.4251, found 849.5634.
HPLC of proteolysis product 15: The 50% B fraction containing
both unprocessed 14 and product 15 was lyophilized and dissolved
in 100 L of 50% B. The solution was injected onto a Varian Micro-
sorb-MV 5
100 Å C18 analytical column (4.6 mm ꢀ 25 cm) and
C
C
82H122N16O19S2 849.4251, found 849.9947; [M+3H]3+ calcd for
l
82H123N16O19S2 566.6192, found 566.9992. UV
e
349 = 18,000 mM
l
ꢁ1 cmꢁ1
.
RP-HPLC applied using a linear gradient from 0% to 70% B over
70 min and a flow rate of 1.0 mL/min. The cleaved product (15) peak
was found at tR = 60.5 min (56% B), ESI-MS (m/z): [M+2H]2+ (av)
calcd for C75H122N12O14S2 740.0028, found 739.9970; [M+3H]3+
(mono) calcd for C75H123N12O14S2 493.2902, found 493.3000. The
starting material (14) eluted at tR = 64.5 min (60% B), ESI-MS (m/z):
[M+2H]2+ (mono) calcd for C91H151N15O17S3 911.0282, found
911.0600; [M+3H]3+ (av) calcd for C91H152N15O17S3 608.1683, found
608.0798.
4.2.2. Synthesis of biotin-K(Dde)KSKTKC(C10BP)K(Dnp)IM, 9
Biotin-K(Dde)KSKTKCK(Dnp)IM,35
(10.0 mg, 5.0 mol,
7
l
1.0 equiv) was dissolved in 2.0 mL of 2:1:1 DMF/n-butanol/0.1%
aqueous TFA (v/v/v) and (3-((((2E,6E)-8-bromo-2,6-dimethylocta-
2,6-dien-1-yl)oxy)methyl)phenyl)(phenyl)methanone, 5 (8.5 mg,
20
5.0 equiv) was dissolved in a small amount 0.1% aqueous TFA
(100 L) and added to the reaction. The reaction was monitored by
RP-HPLC. Following 2 h of reaction time, 200 L of BME was added
lmol, 4.0 equiv), was added. Zn(OAc)2 (5.5 mg, 25 lmol,
l
l
4.3.3. MS-MS analysis of benzophenone-containing substrates
following enzymatic processing by Rce1p
to quench the reaction and reverse any sulfonium salt formation.36
The mixture was stirred for 2.5 h, filtered, diluted with 0.1% aqueous
TFA and purified by RP-HPLC using 0.1% TFA/H2O (solvent A) and
0.1% TFA/CH3CN (solvent B) and the following elution profile per-
formed at 2.5 mL/min: 5.0 min 0% solvent B; linear gradient from
30% to 100% solvent B over 140 min. The solvent mixture was re-
moved by lyophilization to afford 5.3 mg (44%) of 9. Purity by RP-
HPLC: 96.2%, tR = 66 min, ESI-MS (m/z): [M+2Na]2+ calcd for
The lyophilized 40% and 50% B samples containing 10 and 15,
respectively, were dissolved in 30
lL of 100% solvent A (0.1%
TFA/H2O) (10) or 50 L of 50% solvent B (0.1% TFA/CH3CN) (15)
l
and further diluted with an equal volume of 50% CH3CN and 0.1%
formic acid in water to a final concentration of approximately
1
lg/l
L prior to MS analysis. MS was performed by direct injection
(10
l
L) into a QSTAR Pulsar i quadrupole-TOF (time-of-flight) mass
C
115H179N21O28S3Na2 1222.1096, found 1221.8616; [M+2Na+H]3+
spectrometer equipped with a turbo ionspray source, collected as
individual scans for approximately 1 s. The ion spray voltage was
5000 V during MS acquisition and 4500 V during MS/MS with a
collision energy adjusted until the precursor ion selected was
reduced to 10–50% of its peak intensity at 0% collision energy.
The instrument used an external calibration of renin using the
monoisotopic peaks of [M+2H]2+ (m/z = 580.3) (10) and [M+2H]2+
(m/z = 739.4) (15). Mass spectra were the average of scans
collected in positive mode over a 0.2 min acquisition period. MS re-
sults were evaluated with Analyst software and MS/MS fragmenta-
tion predicted using UCSF Protein Prospector v 5.5.0 (http://
prospector.ucsf.edu).
calcd for C115H180N21O28S3Na2 815.0730, found 814.9139;
[M+2Na+2H]4+ calcd for C115H181N21O28S3Na2 611.5548, found
611.4392. UV e .
349 = 18,000 mMꢁ1 cmꢁ1
4.3. Mass spectrometric and HPLC analysis
4.3.1. Large-scale proteolysis reactions for product structure
identification
Stock concentrations of 6 and 14 were diluted in Hepes buffer
(100 mM Hepes, 5 mM MgCl2, pH 7.5) to 40 lM (6) and 20 lM
(14). Rce1p-containing membranes were added to a final concentra-
tion of 0.25 mg/mL (total protein) in a total assay volume of 1–2 mL.
The samples were mixed briefly and incubated in a 35 °C water bath
for approximately 4 h. The crude product was purified using Sep-Pak
reversed phase C18 columns equilibrated in 2 ꢀ 10 mL 0.1% TFA/
CH3CN (solvent B) followed by 2 ꢀ 10 mL 0.1% TFA/H2O (solvent
A). The reaction mixtures containing proteolyzed peptide were ap-
plied to the columns and fractions were eluted using 4–6 mL of sol-
vents A and B with a 10% incremental stepwise gradient from 0% to
100% B at a flow rate of 3–5 mL/min.
The individual fractions were evaluated by ESI-MS, which re-
vealed the presence of doubly- and triply-charged parent ions of
the products of cleavage, 10 and 15, in the 40% and 50% B fractions,
respectively, as well as doubly- and triply-charged parent ions of
the starting materials, 6 and 14, in the 50% B fraction only. These
fractions were lyophilized and re-dissolved in solvent A (6 and
10) or 1:1 solvents A:B (14 and 15) prior to HPLC and MS/MS
analysis.
4.4. Kinetic assay
4.4.1. Rce1p isolation and in vitro fluorescence-based CAAX
proteolysis assay
Carbonate-washed membrane fractions containing yeast Rce1p
were used as the source of enzyme. Membranes were isolated
according to previous methods as 1.0 mg/mL total protein stock
solutions in lysis buffer (50 mM Tris, pH 7.5, 0.2 M sorbitol,
1 mM EDTA, 0.2% NaN3) containing a protease inhibitor cocktail
(1 lg/mL each chymostatin, leupeptin, pepstatin; 0.85 lg/mL apro-
tinin; 1 mM PMSF) and stored at ꢁ80 °C.10,12 The membranes were
diluted with an equal volume of assay buffer (100 mM Hepes, pH
7.5, 5 mM MgCl2) to 0.5 mg/mL and preincubated in a metallic
block for 10 min at 30 °C. Substrates 2, 4, and 6 were serially di-
luted in assay buffer from their stock concentrations (100, 90,
and 90
(three 15
l
M, respectively) into a 96-well V-shaped microtiter plate
L replicates) and transferred to 384-well black flat bot-
l
4.3.2. RP-HPLC analysis of benzophenone-containing substrates
following enzymatic processing by Rce1p
HPLC of proteolysis product 10: The 40% and 50% B fractions
were pooled, lyophilized, and dissolved in 1 mL of 100% solvent A
(0.1% TFA/H2O). The solution was loaded onto a Phenomenex Luna
tom microtiter plate; 10 concentration points for each substrate.
The plate was covered with a lid and incubated 10 min at 30 °C. As-
says were initiated with the addition of 15 lL of diluted mem-
branes to the diluted substrates in the wells, resulting in a final
membrane protein concentration of 0.25 mg/mL, substrate concen-
10
l
100 Å C18(2) preparatory column (10.00 mm ꢀ 25 cm), and
trations ranging from 0–50
lM (2), or 0–45 lM (4 and 6), and a to-
RP-HPLC applied using a linear gradient from 30% to 100% B over
140 min and a flow rate of 2.5 mL/min. The cleaved product (10)
peak denoted by loss of Dnp at 349 nm was found at tR = 28 min
(42% B), ESI-MS (m/z): [M+2H]2+ (mono) calcd for C59H88N10O12S1
580.3146, found 580.3892. The starting material (6) eluted at
tal assay volume of 30 L. The fluorescence in the samples was
l
measured every 30–60 s for 1 h at 30 °C with 5 s shaking before
each reading at 340/420 nm excitation/emission wavelengths
using a BioTek Synergy™ HT microplate fluorometer.

7568
K. Kyro et al. / Bioorg. Med. Chem. 19 (2011) 7559–7569
4.4.2. Kinetic analysis
was used for all samples so that they contained equal quantities
of Rce1p.
Data from experiments was collected as replicates (two for 2
and five for 4 and 6) and analyzed using GraphPad Prism 4.0. Data
was fit to the four parameter Hill Equation Y ¼ Vmax ꢂ Xh=ðKMh þ XhÞ
without constraints after providing approximate initial values.
Prior to analysis, the data (RFUsample) was corrected to take into ac-
count the observation that RFU and product formation are propor-
tional but do not have a 1:1 relationship. In part, this is due to
intermolecular quenching effects that increase as substrate con-
centration increases. A standard curve was created using trypsin
to determine the maximum fluorescence obtainable at various
substrate concentrations (RFUtrypsin). Compounds 2, 4, and 6 all
contain trypsin cleavage sites. A graph of RFUtrypsin versus sub-
strate concentration was plotted for each substrate, and a best-fit
line determined. The equations of the curves were used to extract
the actual product concentration for partially reacted samples
(RFUsample). These values were used for Prism analysis.
4.5.2. Photoaffinity-labeling of Rce1p using biotinylated,
photoactive peptide substrates
Photolysis reactions were conducted for 20 min as described in
Section 4.5. Reactions (67.5
MgCl2, pH 7.5, 15 M photoprobe (11, 12, 13, 14, 8, or 9), and
0.25 g/ L membranes containing or lacking Rce1p. SA pull-down
lL) contained 100 mM Hepes, 5 mM
l
l
l
and Western blotting were performed as described in Section 4.5.1.
4.5.3. Photolysis time course to determine maximal labeling of
Rce1p
Membranes containing Rce1p (0.25
the presence of 14 (15 M) as described in Section 4.5 for varying
amounts of time (10–60 min). All reactions (67.5 L) contained
100 mM Hepes, 5 mM MgCl2, pH 7.5, and 15 M 14. SA pull-down
lg/lL) were irradiated in
l
l
l
and Western blotting were performed as described in Section 4.5.1.
4.5. Photoaffinity labeling
4.5.4. Competition experiments using 2 and 14
All photolysis reactions were conducted at 4 °C in a UV Rayonet
photoreactor (Model # RPR-100, Southern New England Ultraviolet
Co.) equipped with 16 RPR-2537 Å lamps and a circulating plat-
form that allows up to 13 samples to be irradiated simultaneously.
Photolysis reactions were conducted for 30 min as described in
Section 4.5. All reactions (67.5
MgCl2, pH 7.5, 15 M 14, and 0.25
Rce1p. 2 was used at 50 M and 100
l
L) contained 100 mM Hepes, 5 mM
g/ L membranes containing
M in the assays evaluating
l
l l
l
l
All reactions (67.5
tubes (10 ꢀ 43 mm) and contained 100 mM Hepes, 5 mM MgCl2,
pH 7.5, 15 M photoprobe (8, 9, 11, 12, 13, or 14), and 0.25 g/
L membranes containing or lacking Rce1p. The reactions were
l
L) were performed in silanized quartz test
competition. SA pull-down and Western blotting were performed
as described in Section 4.5.1.
l
l
l
Acknowledgments
photolyzed for 30 min (unless otherwise noted) using the appara-
tus described above and quenched by flash-freezing in N2 (l).
Immediately after photolysis and prior to quenching, protease
This research was supported by the National Institutes of
Health Grants GM58442 (M.D.D.) and GM067092 (W.K.S.). The
authors would like to thank Professor George Barany for use of
his automated peptide synthesizer. Some equipment and Bpa
peptides 11–14 were supplied by Edgewood Chemical Biological
Center (ECBC). We thank Bruce Witthuhn for assistance with
acquisition of mass spectral data, LeeAnn Higgins for technical
assistance (both of the University of Minnesota Center for Mass
Spectrometry and Proteomics), and Emily Hildebrandt for impor-
tant discussions.
inhibitor cocktail (Sigma) (1.35 lL) was added to the samples.
4.5.1. Pull-down of photolabeled Rce1p on streptavidin beads
followed by Western Blot analysis
Irradiated samples were thawed and concentrated using protein
precipitation (CalBiochem), solubilized in RIPA buffer [75 lL;
150 mM NaCl, 50 mM TrisꢃHCl, 1.0% (w/v) sodium deoxycholate,
1.0% (v/v) Triton X-100, pH 7.0] containing 1.5% (w/v) SDS and pro-
tease inhibitor cocktail (Sigma) (1:50), and added to spin columns
containing streptavidin agarose resin (0.7 lg of protein/lL resin
Supplementary data
pre-equilibrated with RIPA/1.5% SDS/protease inhibitor cocktail
[1:50]) and incubated 4 h at 4 °C. Following brief centrifugation,
the resin was washed three times with RIPA/1.5% SDS/protease
inhibitor cocktail (Sigma) for 15 min/wash.
Supplementary data (RP-HPLC chromatograms for 6, 9, and 11–
14 are provided as Supplementary data as well as MS/MS sequenc-
ing and comparative summary tables of mass spectral data for 6
and 10. Additional Supplementary data includes MS/MS sequenc-
ing of 11–13, a summary table of mass spectral data for 11–14,
and MS/MS sequencing and comparative summary tables of mass
spectral data for 14 and 15. Photolysis time course data for 14 is
also included) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2011.10.027.
The samples were eluted by adding 2ꢀ sample buffer [20
lL;
4.0% SDS, 20% glycerol (v/v), 125 mM TrisꢃHCl (pH 6.8), 10% BME
(v/v), 0.004% Bromophenol Blue (w/v)] to each spin column, the
eluate heat denatured for 15 min at 70 °C followed by brief centri-
fugation. The samples were separated by SDS–PAGE electrophore-
sis (12% Tris-glycine gels) and transferred to a PVDF membrane.
The PVDF membrane was blocked with 5% (w/v) casein in TBST
[25 mM Tris, 150 mM NaCl, 0.1% Tween-20 (v/v), pH 7.4] for 1 h at
room temperature, then incubated in 2.5% (w/v) casein in TBST
containing anti-HA antibody [mAb HA.11 (HA, 16B12, flu tag),
1:10,000 (Covance)] for 1 h at room temperature. The membrane
was washed with TBST and PBS [137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4] and incubated with 2.5% (w/v)
casein in TBST containing secondary antibody [ECL anti-mouse
IgG, horseradish peroxidase-linked (NA931, from sheep),
1:15,000 (GE Healthcare)] overnight at 4 °C. The membrane was
washed as before and immunodecorated bands visualized using
enhanced chemiluminescence (ECL) with autoradiographic film
followed by densitometric analysis. For all experiments where
the amount of labeling was quantified after pull-down for compar-
ative purposes, the same batch of Rce1p-containing membranes
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