Chemically mediated site-specific cleavage of proteins [9]

Full text of DOI:10.1021/ja0002262

7402
J. Am. Chem. Soc. 2000, 122, 7402-7403
Presently we demonstrate (i) site-specific cleavage of E. coli
dihydrofolate reductase (DHFR)7 analogues containing allylgly-
cine at each of three predetermined sites and (ii) iodine-induced
conversion of a zymogen (rat trypsinogen8,9) to its mature form
(trypsin).
Chemically Mediated Site-Specific Cleavage of
Proteins
Bixun Wang,† Michiel Lodder,† Jia Zhou,†
Teaster T. Baird, Jr.,‡ Kathlynn C. Brown,‡
Charles S. Craik,‡ and Sidney M. Hecht*,†
DHFR analogues having allylglycine (AGly) at predetermined
sites were prepared by in vitro protein synthesis in the presence
of DHFR mRNAs5e having a UAG codon in lieu of the codons
Departments of Chemistry and Biology
UniVersity of Virginia, CharlottesVille, Virginia 22901
Department of Pharmaceutical Chemistry
UniVersity of California
10
for Asp27, Val10, or Glu-1. Inclusion of allylglycyl-tRNACUA
was essential for the synthesis of full length protein (Figure 2),
indicating that allylglycine must be incorporated specifically into
DHFR at the site of each UAG codon.5e
San Francisco, California 94143
The ligand binding and catalytic properties of the DHFR
ReceiVed January 21, 2000
A
analogues were determined.11 Analogues Val10 Gly and Glu-1
AGly had the same specific activity and chromatographic proper-
ties as wild type, while Asp27 AGly was dysfunctional as
anticipated.5e,7 Thus allylglycine could be incorporated into DHFR
at positions -1 and 10 without any obvious effect on ligand
binding capability or catalytic competence (Supporting Informa-
tion).
Treatment of the three DHFR analogues containing allylglycine
with I2 afforded cleavage products having the expected sizes
(illustrated in Figure 3 for Asp27 AGly).12 The cleavage site was
established both by gel and capillary electrophoresis.13 The
cleavage products could be separated readily from unreacted
protein by Ni-NTA chromatography (Figure 3B). While I2-induced
cleavage efficiency was expected to vary from site to site based
on solvent accessibility of the allyl group, protein secondary
structure at the allylglycine site seems to be a more important
determinant of cleavage efficiency (Table 1). Presumably, the ease
of formation of the cyclic intermediate (Figure 1) is critical to
the success of the overall cleavage reaction and certain peptide
conformations (e.g. R-helix) are more conducive to the requisite
cyclization.14
In contrast to native chemical ligation,1 which has defined an
elegant new strategy for protein synthesis via peptide bond
formation at specific sites, there is a paucity of methods for site-
specific cleavage of proteins. The cleavage of (semi)synthetic
proteins at predetermined sites2 would facilitate protein engineer-
ing, and studies of protein structure and function.3 Reported herein
is a novel strategy for cleaving the protein amide backbone at a
single, predetermined site with a simple chemical reagent.
The strategy relies on our recent finding that the 4-pentenoyl
group and certain derivatives can be used as protecting groups
for NR of the aminoacyl moiety in misacylated tRNAs.4 Depro-
tection occurs readily by treatment with aqueous iodine at 25 °C
via a presumed iodolactone intermediate. We reasoned that
analogous cleavage should take place in proteins containing
allylglycine (Figure 1), thus permitting site-specific protein
cleavage. While not normally a protein constituent, allylglycine
is not dramatically different in physicochemical properties than
leucine, isoleucine, and valine; its incorporation into specific sites
in proteins may be envisioned by readthrough of a nonsense
codon5 with a misacylated suppressor tRNA6 activated with
allylglycine.
Allylglycine was also introduced into rat trypsinogen at the
site at which it is normally cleaved to afford mature trypsin.8 I2
* Address correspondence to this author.
† University of Virginia.
(7) (a) Baccanari, D.; Phillips, A.; Smith, S.; Sinski, D.; Burchall, J.
Biochemistry 1975, 14, 5267. (b) Johnson, L. F. In Folates and Pterins;
Blakley, R. L., Benkovic, S. J., Eds.; Wiley: New York, 1984; Vol. 1, p 581.
(c) David, C. L.; Howell, E. E.; Farnum, M. F.; Villafranca, J. E.; Oatley, S.
J.; Kraut, J. Biochemistry 1992, 31, 9813. (d) Lee, H.; Reyes, V. M.; Kraut,
J. Biochemistry 1996, 35, 7012.
(8) The rat trypsinogen used in this study is a variant of native rat
trypsinogen9 having the peptide sequence MGHHHHHHGGGAG in place of
the wild-type activation peptide. A UAG codon was included at position -1,
immediately prior to the authentic trypsin coding region. The hexahistidine
moiety facilitated purification of the derived trypsinogen analogue on Ni-
NTA agarose. (Janknecht, R.; de Martynoff, G.; Lou, J.; Hipskind, R. A.;
Nordheim, A.; Stunnenberg, H. G. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
8972.)
‡ University of California.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776. (b) Liu, C.-F.; Tam, J. P. J. Am. Chem. Soc. 1994, 116, 4149.
(2) Lactic acid (e.g., Fahnestock, S.; Rich, A. Science 1971, 173, 340) and
glycolic acid (Chung, H.-H.; Benson, D. R.; Schultz, P. G. Science 1993,
259, 806) can be incorporated into proteins at discrete sites, affording base-
labile ester bonds, but the derived protein analogues contain at least one linkage
that can alter the stability and enzymatic activity of the protein (Chapman,
E.; Thorson, J. S.; Schultz, P. G. J. Am. Chem. Soc. 1997, 119, 7151).
(3) A limited number of cleavage reagents have been reported; cleavage
typically occurs at defined sequence but multiple sites (see, e.g.: (a) Lawson,
W. B.; Gross, E.; Foltz, C. M.; Witkop, B. J. Am. Chem. Soc. 1961, 83, 1509.
(b) Hotez, P. J.; Trang, N. L.; McKerrow, J. H.; Cerami, A. J. Biol. Chem.
1985, 260, 7343. (c) Marcello, A.; Loregian, A.; De Filippis, V.; Fontana,
A.; Hirst, T. R.; Palu`, G. FEMS Microbiol. Lett. 1996, 136, 39).
(4) (a) Lodder, M.; Golovine, S.; Hecht, S. M. J. Org. Chem. 1997, 62,
778. (b) Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V. A.; Hecht,
S. M. J. Org. Chem. 1998, 63, 794.
(9) (a) Craik, C. S.; Largman, C.; Fletcher, T.; Roczniak, S.; Barr, P. J.;
Fletterick, R.; Rutter, W. J. Science 1985, 228, 291. (b) Graf, L.; Craik, C,
S.; Patthy, A.; Roczniak, S.; Fletterick, R. J.; Rutter, W. J. Biochemistry 1987,
26, 2616. (c) Perona, J. J.; Hedstrom, L.; Wagner, R. L.; Rutter, W. J.; Craik,
C. S.; Fletterick, R. J. Biochemistry 1994, 33, 3252.
(5) (a) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G.
Science 1989, 244, 182. (b) Bain, J. D.; Diala, E. S.; Glabe, C. G.; Wacker,
D. A.; Lyttle, M. H.; Dix, T. A.; Chamberlin, A. R. Biochemistry 1991, 30,
5411. (c) Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature
1992, 356, 537. (d) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 621. (e) Karginov, V. A.; Mamaev, S. V.; An, H.;
Van Cleve, M. D.; Hecht, S. M.; Komatsoulis, G. A.; Abelson, J. N. J. Am.
Chem. Soc. 1997, 119, 8166. (f) Short, G. F., III; Golovine, S. Y.; Hecht, S.
M. Biochemistry 1999, 38, 8808.
(6) (a) Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem.
1978, 253, 4517. (b) Heckler, T. G.; Zama, Y.; Naka, T.; Hecht, S. M. J.
Biol. Chem. 1983, 258, 4492. (c) Heckler, T. G.; Chang, L.-H.; Zama, Y.;
Naka, T.; Chorghade, M. S.; Hecht, S. M. Biochemistry 1984, 23, 1468. (d)
Baldini, G.; Martoglio, B.; Schachenmann, A.; Zugliani, C.; Brunner, J.
Biochemistry 1988, 27, 7951. (e) Bain, J. D.; Glabe, C. G.; Dix, T. A.;
Chamberlin, A. R.; Diala, E. S. J. Am. Chem. Soc. 1989, 111, 8013. (f)
Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz,
P. G. Nucleic Acids Res. 1989, 17, 9649. (g) Noren, C. J.; Anthony-Cahill, S.
J.; Suich, D. J.; Noren, K. A.; Griffith, M. C.; Schultz, P. G. Nucleic Acids
Res. 1990, 18, 83. (h) Hecht, S. M. Acc. Chem. Res. 1992, 25, 545.
(10) Allylglycyl-tRNACUA was prepared and characterized in analogy with
other misacylated tRNAs (Supporting Information).6 To demonstrate the
versatility of this misacylated tRNA, substitution at positions 10 and 27 in
DHFR was carried out using rabbit reticulocyte lysate,5e while Glu-1-AGly
was elaborated in an E. coli S30 system.5f
(11) Catalytic competence was determined by oxidation of NADPH
(monitored by the decrease in absorption at 339 nm).7a The ability of DHFR
analogues to bind to Ni-NTA agarose and methotrexate-agarose was used to
determine whether each analogue (i) contained a hexahistidine moiety at the
N terminus and (ii) folded in a fashion similar to wild type, respectively.
(12) The optimal iodine concentration for protein cleavage differed slightly
among DHFR analogues containing AGly at different positions (Table 1). Wild-
type DHFR, and analogues Asp27Val and Glu-1Val gave no cleavage products
under the same conditions (Table 1).
(13) For DHFR analogue Glu-1AGly, I2 treatment afforded a protein that
comigrated with an authentic standard by high-resolution PAGE and native
capillary electrophoresis.
(14) The actual distances between the relevant carboxamide backbone
residues and side chain carbon atoms determined crystallographically for
DHFR support this interpretation (data not shown).
10.1021/ja0002262 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/12/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 30, 2000 7403
Figure 1. Scheme illustrating how treatment of an allylglycine-containing protein with I2 can lead to site-specific cleavage of the protein backbone.
Table 2. Comparison of Kinetic Parameters Obtained from
Trypsin Generated by Iodine Treatment of Trypsinogen Containing
Allylglycine (AGly-Tg) and Wild-type Trypsin (Tna)
sample
kcat (S-1
)
KM (µM)
12 ( 2
kcat/KM (µM-1 s-1
)
Tna
44.1 ( 1.6
3.7 ( 0.8
4.0 ( 0.4
3.0 ( 0.5
3.5 ( 0.7
3.7 ( 0.5
Tn + Lb
42.3 ( 1.3 10.5 ( 1.4
42.5 ( 2.4 14.1 ( 2.8
41.2 ( 2.7 11.6 ( 2.9
Tn + L + THF
Tn + L + THF + I2
Figure 2. Autoradiogram of a 15% SDS-polyacrylamide gel illustrating
the in vitro synthesis of DHFR analogues containing allylglycine at
predetermined sites. Protein synthesis5 was carried out using [35S]-
methionine with wild-type mRNA (lane 1), or mRNA containing UAG
at position 27 (lanes 2 and 3) or position 10 (lanes 4 and 5) and suppressor
tRNAs as noted. Lane 1, no suppressor tRNA; lane 2, unacylated
tRNACUA; lane 3, allylglycyl-tRNACUA; lane 4, unacylated tRNACUA; lane
5, allylglycyl-tRNACUA. Suppression efficiencies are shown below each
lane; the yield of wild type was approximately 20 µg/mL of incubation
mixture.
AGly-Tg + THF + I2 42.1 ( 1.7 11.4 ( 1.6
a Synthesized in vivo as rat trypsinogen and subsequently activated
with enterokinase (1:20, w/w). Active Tn was purified by sequential
hydrophobic and affinity column chromatography methods. b Wild-type
Tn was combined with rabbit reticulocyte lysate (L) and subjected to
the same treatment used to prepare AGly-Tg.
Wild-type trypsinogen and trypsinogen analogues containing
either
allyl-
glycine or valine at the normal cleavage site were assayed for
trypsin activity,15 before and after I2 treatment. As shown (Table
2), only the allylglycine-containing trypsinogen could be activated
with I2. The kinetic parameters of this trypsin were measured and
were comparable to that of wild type. The presence of trypsin
activity following iodine treatment supports the chemical mech-
anism proposed in Figure 1, since a free N-terminal amino group
in trypsin at the authentic cleavage site16 is a prerequisite for
enzymatic activity.17 Additionally, a synthetic tripeptide having
the same sequence as trypsinogen analogue AGly-Tg at the
putative cleavage site (GlyAGlyIle) was shown explicitly to
undergo the anticipated cleavage reaction in good yield (Sup-
porting Information).
These experiments establish the feasibility of a novel strategy
for site-specific cleavage of protein backbones by elaboration of
proteins containing allylglycine at predetermined sites. The
activation of trypsin from trypsinogen by I2 treatment is the first
report of activation of a zymogen by a chemical treatment; this
approach has potential utility for the study of time-dependent
processes mediated by trypsin or other proteins having inactive
precursors. Other possible applications include the systematic
removal of individual protein domains of putative higher order
structures.
Figure 3. Iodine-induced cleavage of DHFR analogue Asp27AGly. The
purified protein was treated with I2 in 100:15 water-THF at 25 °C for
30 min. Panel A: lane 1, Asp27 AGly without I2 treatment; lane 2, Asp27
AGly + 1.0 mM I2; lane 3, Asp27 AGly + 1.5 mM I2; lane 4, wild-type
DHFR + 1.5 mM I2; lane 5, wild-type DHFR without I2 treatment. Panel
B: Asp27 AGly was treated with 1.2 mM I2 (lane 1), then applied to a
Ni-NTA column. The (uncleaved) protein that was retained by the column
was analyzed in lane 2; the nonretained cleavage product appears in lane
3. Cleavage efficiencies are shown below each lane. The N-terminal
peptide cleavage product could not be observed, plausibly due to
peptidases present in the protein-synthesizing system.
Table 1. Iodine-Induced Backbone Cleavage Efficiency of DHFR
Analogues with Allylglycine (AGly) Located in Different Secondary
Structure Contexts
optimal [I2] optimal
secondary structure solvent
DHFR
analogue
for cleavage cleavage in which AGly was
acces-
(mM)
eff (%)
incorporated
sibilityb
Acknowledgment. We thank Dr. James Landers and Braden Giordano
for assistance with capillary electrophoresis. This work was supported at
the University of Virginia by Research Grant CA77359 from the National
Cancer Institute and at UC San Francisco by Research Grant MCB9604379
(C.S.C.) and by a fellowship (T.T.B.) from the National Science
Foundation.
wild type
nda
62
11
Asp27AGly
Val10AGly
Glu-1AGly
Glu-1Val
1.2
2.0
1.5
R-helix
+
hydrogen bonded turn ++
41
extended strandb
+++
+++
nda
a Not detectable. b Inferred from the crystal structure of DHFR,7c,d
assuming that the modified protein structures are fundamentally
analogous to those of wild type. Increased solvent accessibility is
denoted by increasing numbers of pluses.
Supporting Information Available: Experimental details of protein
preparation, cleavage, and assay (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JA0002262
treatment should thus result in the chemical activation of trypsin
from its inactive precursor. In common with wild-type rat
trypsinogen, the in vitro synthesized trypsinogen containing AGly
(AGly-Tg) had similar minimal tryptic activity. However, the
trypsinogen containing AGly could not be converted to active
trypsin through proteolysis mediated either by enteropeptidase
or trypsin since the zymogen activation sequence was not present.8
(15) Zimmerman, M.; Ashe, B.; Yurewicz, E. C.; Patel, G. Anal. Biochem.
1977, 78, 47.
(16) Pasternak, A.; Liu, X.; Lin, T.-Y.; Hedstrom, L. Biochemistry 1998,
37, 16201.
(17) (a) Scrimger, S. T.; Hofmann, T. J. Biol. Chem. 1967, 242, 2528. (b)
Huber, R.; Bode, W. Acc. Chem. Res. 1978, 11, 114.
(18) Varshney, U.; Lee, C.-P.; RajBhandary, U. L. J. Biol. Chem. 1991,
266, 24712.