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INTRODUCTION |
All natural proteins known so far are linear chains of amino acids
that fold into a unique three-dimensional structure dictated by the
sequence of amino acids. A cyclic backbone structure has been found and
synthetically introduced in small peptides, but is an almost unexplored
topic in protein chemistry and protein structural research, in
particular for larger proteins. The circular topology is also expected
to lead to improved stability due to the reduced conformational entropy
in the denatured state, according to polymer theory (1). It has indeed
been shown experimentally that cyclized
-lactamase was stabilized
against heat precipitation and exopeptidase degradation (2) and that
the cyclization also improved in vitro thermal stability of
dihydrofolate reductase (3). Although there was no significant
stabilization observed in the pioneering work on a cyclic bovine
pancreatic trypsin inhibitor prepared by chemical modification, several
effects may have canceled out in this example (4).
A recent development in protein chemistry, the use of self-splicing
proteins (often called inteins), has opened a general avenue to create
a circular backbone topology as well as to ligate proteins and peptides
in vitro. This procedure has been called expressed protein
ligation or intein-mediated protein ligation (Refs. 5 and 6 and, for
review, see Ref. 7). In this approach, an intein with an
asparagine-to-alanine mutation in the active site is fused to one
fusion partner. This mutation stops the enzymatic reaction at the stage
of a C-terminal thioester of the fusion partner, selectively cleavable
by thiols. The second peptide carrying an N-terminal cysteine acts as
an S-nucleophile at the thioester group, forming a new
peptide bond after S-N rearrangement. However, this approach has the
disadvantage that it requires the nucleophilic thiol group of cysteine
at the N terminus of one partner as well as a C-terminal thioester
modification of the other partner, whose formation is catalyzed by the
intein (5, 6). This reaction can be used to make cyclic peptides and
proteins, but the intramolecular cyclization reaction always has to
compete with other intermolecular reactions such as polymerization and hydrolysis of the thioester group (2, 8, 9), which reduce the
cyclization efficiency and complicate the purification procedure (2).
An in vivo cyclization without by-products would be of
importance for the large-scale production of cyclic proteins and
peptides in vivo. This may also provide the possibility for
creating a large library of cyclic peptides and proteins in
vivo for functional selections as well as for biophysical
characterization of cyclic proteins. Two independent groups have
demonstrated that the naturally split intein DnaE from
Synechocystis sp. PCC6803 (Ssp DnaE)
can be used for cyclization of proteins and peptides in vivo
by arranging the naturally separated two fragments of the intein,
DnaEN (the N-terminal part) and DnaEC (the
C-terminal part), in the order DnaEC-extein-DnaEN (3, 10). In theory, this
strategy should be feasible not only with the naturally split intein,
but also with any intein by artificially creating a similar
arrangement. This corresponds essentially to a circular permutation of
a precursor protein that introduces new N and C termini into the intein
domain. The end products should include a circular protein if the
precursor protein folds into a functional structure (see Fig. 1).
In this report, we have tested our idea of cyclization by
circular permutation of precursor proteins within intein domains using
the intein PI-PfuI1 from Pyrococcus
furiosus. We chose the green fluorescent protein (GFP) as our cyclization target because
cyclic GFP might become a useful tool for studies on the roles of N and
C termini in cellular environments.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction for Circular GFPuv Expression--
The
C-terminal part (PI-PfuIC, residues 161-454) of
the intein PI-PfuI was amplified from pHisIn
(11) using
oligonucleotides 5'-CTTTAAGAAGGAGATATA-3' and
5'-CTCAGTAAGAGATTTTTTTCTAGATCCGGTGTTGTGGACG-3', followed by digestion
with NcoI and XbaI. The N-terminal part (PI-PfuIN, residues 1-160) of the intein was
amplified from pCBD
In (11) using oligonucleotides
5'-GTTCGGTACCGGATGCATAGACGGAAAGG-3' and
5'-TGGAAGCTTACTTAACATGTGAGTGG-3', followed by digestion with KpnI and HindIII. The plasmids pHisIn
and
pCBD
In were kindly provided by Dr. T. Yamazaki (Osaka University).
The gene of GFP, improved by DNA shuffling (GFPuv, also called the
Cycle3 mutant) and bearing the mutations F99S, M153T, and V163A (12),
was obtained from pBAD/AC2 by polymerase chain reaction (PCR)
amplification with primers
5'-CCTCTAGACATCATCACCACCATCACTCTAGAAAAGGAGAAGAACTYTTC-3' and
5'-TAGGTACCGCGTGGCACCAACCCAGCAGCWGTTAC-3'. The polymerase chain
reaction product was digested with XbaI and
KpnI. These three PCR products were then ligated to form a
fusion protein under the control of the T7 promoter in the expression
vector pTFT74 (13) between the NcoI and HindIII
sites in a stepwise manner, yielding the plasmid pIWT5563his.
Expression and Purification--
For protein expression,
Escherichia coli strain BL21-CodonPlus(DE3)-RIL (Stratagene)
was transformed with plasmid pIWT5563his. The use of this strain, which
overexpresses E. coli tRNAs for rare codons, was necessary
due to the large number of rare codons in the PI-PfuI gene.
The cells were grown at 30 °C in 1 liter of LB medium containing 50 µg/ml ampicillin and 30 µg/ml chloramphenicol until
A550 ~ 0.5 was reached. The protein was then
expressed at a slow rate by incubation at room temperature
(~25 °C) overnight without induction, taking advantage of the
leaky expression vector, followed by addition of
isopropyl-
-D-thiogalactopyranoside to a final
concentration of 0.1 mM and incubation for another 6 h. The cells were harvested by centrifugation at 4000 × g for 10 min at 4 °C. The cell pellets, which showed
bright green fluorescence, were resuspended in 50 mM sodium
phosphate buffer (pH 8.0) and 300 mM NaCl. The cells were
lysed by incubation with lysozyme (1 mg/ml) for 30 min and by
sonication on ice. The cell lysate was loaded on a 10-ml
Ni2+-NTA column (QIAGEN Inc.) after centrifugation at
20,000 × g for 1 h at 4 °C. The column was
washed with 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 20 mM imidazole. The
hexahistidine-tagged proteins including GFPuv were eluted with 300 mM imidazole (see Fig. 3B, lane 2).
The eluted sample was further purified by organic extraction as
described by Yakhnin et al. (14). Briefly, the sample was
saturated with ammonium sulfate and extracted with ethanol. The aqueous
phase was subsequently removed from the organic phase after addition of
1-butanol. The extracted aqueous fraction contained only a single band
of ~28 kDa (see Fig. 3B, lane 3). The linear
form of GFPuv was prepared by digestion of the circular form with
thrombin. For this purpose, 1.5 ml of 10 µM cyclic GFPuv was incubated with 0.2 units of thrombin (Roche Molecular Biochemicals) for 2.5 h at room temperature, followed by purification on a 10-ml Ni2+-NTA column. The single band of the purified linear
protein migrated more slowly than the circular form of GFPuv (see Fig.
3B, lane 4).
To examine the splicing reaction in vivo, the E. coli cells containing plasmid pIWT5563his were grown
overnight at room temperature in LB medium, followed by induction with
a final concentration of 0.1 mM
isopropyl-
-D-thiogalactopyranoside for 4 h, and
then either directly mixed with SDS loading buffer and boiled at
95 °C for 5 min or alternatively first sonicated and then
resuspended in SDS loading buffer. The cell lysates were separated by
12% SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene difluoride membrane for Western blot analysis. Western blotting was performed using the Tetra·His AntibodyTM (QIAGEN Inc.) and an anti-GFP antibody (Roche
Diagnostics) and visualized by colorimetric detection using alkaline
phosphatase-conjugated anti-mouse IgG.
The structure of cyclic GFP was modeled based on the crystal structure
of GFP (Protein Data Bank code 1EMC) (15) as follows. The
modeled structure was obtained by simulated annealing using artificial
distance constraints created from the crystal structure based on
hydrogen bonds as well as proton-proton distances within 5 Å. The
distance constraints between hydrogen atoms for the model building were
artificially created as upper distance constraints based on
proton-proton distances of <5 Å of all the hydrogen atoms calculated
in the crystal structure with the program MOLMOL (16). All the methyl
and methylene groups were treated as pseudo atoms. The hydrogen bonds
found in the crystal structure were used as upper and lower distance
constraints for the structure calculation. The chromophore structure
was treated as the original unmodified amino acids during the
calculation. The covalent bond between the N and C termini was
introduced as upper and lower distance constraints. For the linker
connecting both termini, there was no constraint applied besides the
covalent bond connecting the termini. With a total of the 9188 distance
constraints, obtained from both proton-proton distances and hydrogen
bonds, three rounds of simulated annealing calculation were performed
to obtain the model structure depicted in Fig. 2B. All
calculations were performed with the program DYANA (17) on a SGI Octane.
Circular Dichroism Measurements--
CD wavelength scans were
recorded with a Jasco J-715 spectropolarimeter. The protein samples
were prepared in 100 mM potassium phosphate (pH 8.0), 50 mM NaCl, and 1 mM EDTA; the protein
concentrations were 0.12 mg/ml (linear) and 0.15 mg/ml (circular). All
spectra were recorded at 25 °C with 10 scans. The data were
normalized to molar ellipticity with a path length of 0.1 cm.
Fluorescence Spectroscopy of the Cyclized and Linear Forms of
GFPuv--
All fluorescence spectra were recorded with a PTI
Alpha Scan spectrofluorometer at 25 °C. A cuvette with 1-cm path
length was used. The samples for the emission and excitation scans were prepared in 50 mM potassium phosphate (pH 6.5, 7, 7.5, or
8), 1 mM EDTA, and 1 mM DTT or in 50 mM Tris (pH 8.5), 1 mM EDTA and 1 mM DTT. The final protein concentration of all the samples
was 1 µg/ml. For the excitation spectrum, the fluorescence intensity was monitored at 508 nm, whereas the emission spectrum was recorded with an excitation at 397 nm.
Unfolding and Refolding Kinetic Measurements--
The kinetic
unfolding and refolding experiments were performed in solutions
containing 50 mM potassium phosphate (pH 8), 50 mM sodium chloride, 1 mM EDTA, 1 mM
DTT, and various concentration of GdnHCl. In the unfolding experiments,
the protein solution was diluted 1:320 in the unfolding solution
containing GdnHCl, resulting in a final protein concentration of 0.5 µg/ml at 25 °C. The change in the chromophore fluorescence
intensity was fitted with a single exponential,
A·exp(
k·t), where A,
k, and t are amplitude, kinetic constant, and
time, respectively, using SigmaPlot software (SPSS Inc., Chicago, IL).
This equation assumes no residual fluorescence at infinite time. In the
refolding experiments, the proteins were first denatured in 6 M GdnHCl for 3 h at room temperature (see Fig.
6B) (18, 19) or denatured in 7 M GdnHCl for
various times at room temperature (see Fig. 7), followed by 1:25-40
dilution in the refolding buffer. The final protein concentration was
~0.5 µg/ml for all measurements. The change in the chromophore
fluorescence intensity was fitted with a double exponential,
A·(1
exp(
k1·t)) + B·(1
exp(
k2·t)) + C, where
k1 and k2 are the kinetic
constants for the fast and the slow phases, respectively. The fraction
of the fast phase in Fig. 7C was determined by calculating
A/(A + B) from the fitted values. The
concentrations of GdnHCl were determined after the kinetic measurements
by measuring the refractive index.
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RESULTS |
Design of the Construct for in Vivo Cyclization--
Our concept
to produce a circular protein in vivo is simple. The
conventional and natural protein splicing activity of an intein is to
connect the C terminus of the N-terminal fragment (N-extein) and the N
terminus of the C-terminal fragment (C-extein) (Fig.
1A). These two interacting
termini can also be within one extein domain. Topologically, this
corresponds to joining the original N and C termini of the extein
domains and, at the same time, introducing new termini into the intein
domain (Fig. 1B). This is nothing but a circular permutation
of the precursor protein.

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Fig. 1.
Schematic representation of the circular
permutation approach for backbone cyclization. A,
ligation of two extein domains by protein splicing; B,
circular permutation of the precursor protein containing an intein
domain. Protein splicing results in three fragments, including an
extein with a circular peptide backbone.
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After the protein splicing reaction, the precursor protein gives rise
to three protein fragments, viz. a cyclized extein and the
N- and C-terminal parts of the intein. For our study, we chose the
intein PI-PfuI from P. furiosus because it has
been demonstrated that this intein can be artificially split in the
extended loop between residues 160 and 161 to perform
trans-ligations in vitro (11, 20, 21).
As the target for cyclization (i.e. the extein), we chose
the improved green fluorescent protein GFPuv (also called Cycle3 mutant) (12) since cyclic GFP might constitute a new tool for studying
the roles of N or C termini or protein backbone topology in cellular
processes such as protein degradation and translocation in
vivo. Moreover, the properly folded end product can be
conveniently monitored by its green fluorescence. Furthermore, GFP had
been successfully circularly permuted by joining the N terminus
(residue 1) and the C terminus (residue 238) with a linker of 6 amino
acids (22, 23), suggesting that a new connecting loop would likely lead
to functional GFP.
The crystal structure of GFP indicates that this protein has a
cylinder-like
-barrel structure containing the chromophore in the
distorted central helix surrounded by the
-barrel (Fig. 2B) (15, 24, 25). The visible
N and C termini are not in very close proximity (~19 Å between
residues 2 and 229), but are located on the same side of the cylinder.
The N-terminal residue and the C-terminal fragment after residue 229 in
the crystal structures of GFP are often invisible and probably
disordered (15, 24, 25). The original sequence of GFPuv from residues 3 to 228 is unchanged in our construct (Fig. 2A,
underlined), and a hexahistidine tag was added in front of
the GFPuv gene, whereas a thrombin-specific proteolytic site (LVPRGT,
similar to the thrombin-specific sequence LVPRGS, with the Thr residue
being introduced to generate a KpnI site) was introduced
behind the GFPuv gene (Fig. 2A). The hexahistidine tag and
its flanking sequence together constitute an 11-residue linker between
the N-terminal intein domain (PI-PfuIC) and the second residue of GFPuv. The thrombin recognition sequence is part of a
6-residue linker between Leu229 of GFPuv and the C-terminal
intein domain (PI-PfuIN).

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Fig. 2.
The construct for in vivo
cyclization of GFPuv. A, the detailed construct
of the precursor protein containing an artificially split intein
(PI-PfuI) and an extein (GFPuv). The original amino acid
sequence of GFPuv is underlined. The hexahistidine tag in
front of GFPuv (in boldface) and the thrombin recognition
site are shaded. The thrombin cleavage site is indicated by
an arrowhead. The original terminal residue numbers of
GFPuv, between which three-dimensional structure is visible, are
labeled on top of the primary sequence. The first N-terminal residue of
the extein domain is in italics. B, model of
cyclized GFPuv from the crystal structure of a GFP calculated by the
program DYANA using artificial constraints obtained from the x-ray
structure (Protein Data Bank code 1EMC) (see "Experimental
Procedures"). The original N- and C-terminal locations are indicated
by N and C, respectively. The first N-terminal
residue of the extein part is displayed in a ball-and-stick model. The
chromophore is shown in a Corey-Pauling-Koltun model. The figure was
created with the program MOLMOL (16).
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Fig. 2B provides an overview of the modeled cyclic GFPuv
structure, and the first residue (threonine) after the N-terminal intein domain (PI-PfuIC) is depicted as a
ball-and-stick model. The distance between residues 2 and 229 is ~19
Å. The linker of 17 amino acids, resulting from the spliced N- and
C-terminal extension, should be more than sufficient to connect
residues 2 and 229. Even an extended linker of 5 or 6 residues might be
sufficiently long to span this distance. However, we have observed that
the removal of 8 residues including the hexahistidine tag resulted in
weaker fluorescence of the cells (data not shown). This is probably a
consequence of the linker between PI-PfuIC and
GFPuv being too short to allow efficient folding into the correct
structure of both GFPuv and the intein domains due to some steric
hindrance. In this study, we have focused on the GFPuv construct of 245 amino acids shown in Fig. 2A. The presence of residue 229 (isoleucine) was found to be essential for functional structure
formation of GFP in a truncation experiment, but it could be replaced
by leucine with no measurable loss of fluorescence (26). Therefore, we replaced this residue with leucine to introduce a thrombin-specific proteolytic site.
GFPuv-extein and the artificially split inteins together form a
precursor protein with a total length of 720 amino acids (83 kDa). Upon
protein splicing, it is expected that three fragments are released: the
N-terminal fragment PI-PfuIC (315 amino acids, 37 kDa) with an N-terminal hexahistidine tag, the C-terminal fragment containing PI-PfuIN (160 amino acids, 18.7 kDa)
without any tag, and cyclized GFPuv (245 amino acids, 27.5 kDa) with an
internal hexahistidine tag and a thrombin cleavage site.
In Vivo Cyclization of GFPuv and Purification of Cyclic
GFPuv--
The precursor protein consisting of the intein
domains and GFPuv was expressed in LB medium at room temperature as
described under "Experimental Procedures." The cells were harvested
and showed bright green fluorescence under a UV lamp of 365-nm
wavelength. Total cell lysates were prepared either by resuspension in
SDS loading buffer containing 1 mM DTT and boiling at
95 °C for 10 min (Fig. 3A,
lanes 2 and 5) or by sonication in 20 mM Tris (pH 8.0) and resuspension in SDS loading buffer
containing 1 mM DTT, followed by boiling at 95 °C for 5 min (lanes 3 and 6). The cell lysates were
analyzed by Western blotting with an anti-GFP antibody (Fig.
3A, lanes 2-4) and an anti-His tag antibody
(lanes 5-9). Two stronger bands of ~80 and 28 kDa were
detected with the anti-His tag antibody as well as with the anti-GFP
antibody, corresponding to the precursor protein and GFPuv. The two
samples prepared by sonication or by boiling showed the same pattern on
the Western blots, indicating that the ~30-kDa bands were not
produced during cell disruption (Fig. 3A). The 28-kDa band
migrated more slowly after thrombin digestion and at the same position
as the linear form, as explained in detail below, strongly suggesting
that this band is the circular form of GFPuv, which is
present in vivo already before purification (Fig.
3A, lanes 4 and 7). It is noteworthy that there was no detectable band on Western blots at the position of
purified linear GFPuv, but we do not know the nature of the ~33-kDa
band, which might be a proteolytically truncated precursor (Fig.
3A, lanes 2 and 3).

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Fig. 3.
SDS-polyacrylamide gel electrophoresis
analysis of in vivo cyclization in E. coli.
A, Western blot analysis. Lanes 2-9 are Western
blots using either an anti-GFP antibody (lanes 2-4) or an
anti-His tag antibody (lanes 5-9). Lane 1, total
cell lysate prepared by boiling and stained with Coomassie Blue;
lane 2, total cell lysate prepared by boiling; lane
3, total cell lysate prepared by sonication; lane 4,
total cell lysate prepared by sonication and digested with thrombin;
lane 5, total cell lysate prepared by boiling; lane
6, total cell lysate prepared by sonication; lane 7,
total cell lysate prepared by sonication and digested with thrombin;
lane 8, purified linear GFPuv; lane 9, purified
cyclic GFPuv. B, purification. Lane 1,
supernatant of the cell lysate; lane 2, elution from a
Ni2+-NTA column as described under "Experimental
Procedures"; lane 3, after organic extraction; lane
4, after thrombin digestion. All samples in B were
analyzed on Coomassie Blue-stained 12% SDS-polyacrylamide gels.
lin-GFP, linear GFPuv; cyc-GFP, cyclic
GFPuv.
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After the protein was expressed on a large scale (1 liter), the cell
lysate was centrifuged, and the soluble fraction was applied to a
Ni2+-NTA column. Bound proteins were eluted with 300 mM imidazole (Fig. 3B, lane 2). There
were four major bands of 80, 35, 28, and 19 kDa in the eluted fraction.
The band with the highest molecular mass corresponds to the unspliced
precursor protein. The product of 28 kDa was identified as GFPuv by a
second step of purification and mass spectrometry. The 35- and 19-kDa
fragments are likely to be the spliced and noncovalently associated N-
and C-terminal intein domains PI-PfuIN and
PI-PfuIC, respectively, because they could
associate under nondenaturing conditions to form the whole functional
intein, and then both were purified via the N-terminal hexahistidine
tag of PI-PfuIC (cf. the 35-kDa band
in the anti-His tag blot) (Fig. 3A, lanes 5 and
6). It is worth mentioning that elution from the
Ni2+-NTA column resulted in a bright green fluorescent
fraction, indicating the presence of properly folded GFPuv. To purify
GFPuv further, the elution fraction was subjected to organic solvent
extraction (14). After the extraction, there was only a single band of 28 kDa (Fig. 3B, lane 3), and the yield was ~3
mg from 1 liter of bacterial culture. The solution had a bright green
color, and the ratio of absorbance at 397 nm to that at 280 nm was
~1.2, which is a good indication of the purity of GFP (27).
Furthermore, there were no detectable polymeric forms found.
We have shown that this single band is indeed the cyclized form of
GFPuv (see "Confirmation of Cyclization of GFPuv" below). It
should be emphasized that there was no detectable contaminant at the
position of the purified linear form, neither in the elution of the
Ni2+-NTA column nor in the organic extraction fraction.
This could be an advantage over other in vitro cyclization
methods, which always involve a mixture of linear and circular forms
(2, 8, 9). Even the in vivo cyclization using the naturally
split intein Ssp DnaE yielded a fraction of the linear form
(3, 10). We have observed the unspliced full-length precursor protein
in the cell extracts, but no significant linear form of GFPuv was present in the purified fraction. To regenerate the splicing activity, the precursor protein has to fold correctly. Once properly folded into
the correct structure, the splicing efficiency is probably high. Since
it might be more difficult to form the functional structure of the
artificially split intein than that of the naturally split intein, the
precursor might be more aggregation-prone, and only correctly spliced
molecules might escape precipitation.
Confirmation of Cyclization of GFPuv--
The cyclization was
confirmed by electron spray ionization mass spectrometry and Edman
degradation after linearization by thrombin cleavage. The target
protein was designed to have a unique and specific proteolytic site
(LVPRGT, similar to the normal thrombin recognition sequence LVPRGS)
following the GFPuv sequence and in front of the
PI-PfuIN sequence (Fig. 2A). Purified
GFPuv was treated with thrombin, followed by amino acid sequencing. We
obtained the sequence GTGTG for the digested linear protein, but
no sequence was detected for the purified GFPuv sample without thrombin
digestion, confirming that the cyclization was successful. The
precursor would have given the sequence GTGCI (Fig. 2A). The
purified protein migrated faster than the linear protein upon
SDS-polyacrylamide gel electrophoresis, which is also expected for
circular proteins because the circular form would have a smaller radius
of gyration than the linear form in the denatured state.
The molecular mass of purified GFPuv before and after the thrombin
treatment was analyzed by electron spray ionization mass spectrometry
(Fig. 4). From the amino acid sequence,
the unmodified linear form is expected to have a molecular mass of
27,537.8 Da. Chromophore formation is due to an autocyclodehydration
(
H2O) and an autooxidation (
2H); therefore, linear
GFPuv with the properly formed chromophore is expected to lose 20 Da
and to have a molecular mass of 27,517.8 Da (28). The observed
molecular mass of the non-thrombin-treated sample was, however,
27,498.4 ± 1.5 Da, which is 19 Da smaller than the expected value
for the linear molecule, indicating loss of one water molecule. This is
consistent with cyclization (dehydration) (Fig. 4). Subsequent specific
proteolysis with thrombin increased the mass to
27,518.8 ± 2.0 Da, suggesting hydration (18 Da), viz.
hydrolysis of a peptide bond. These observations fit perfectly with the
cyclization of this molecule and, at the same time, with the proper
formation of the chromophore, which can also be observed by its green
fluorescence.

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Fig. 4.
Electron spray ionization mass spectrometry
of purified GFPuv. The thick line is the spectrum of
purified GFPuv. The thin line is the spectrum of the sample
after proteolytic digestion by thrombin. Linear GFPuv with the properly
formed chromophore is expected to have a molecular mass of 27,517.8 Da.
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Comparison of Fluorescence and CD Spectra of the Cyclic and Linear
Forms of GFPuv--
The fluorescence spectrum of GFP is a good
indicator of the correctly folded structure of GFP because the unique
fluorescence of GFP is a result of both its chromophore formation and
properly folded three-dimensional structure. In Fig.
5B, the fluorescence properties of cyclized GFPuv and thrombin-linearized GFPuv are compared. Both the emission and excitation profiles of the cyclic and
linear forms of GFPuv are essentially identical, which strongly suggests that the three-dimensional structures of both forms are also
very similar because the fluorescence of the chromophore is sensitive
to its structural environment (28). There were no differences in the
fluorescence properties observed between the circular and linear forms
of GFPuv at pH 6.0-8.5 (data not shown). The emission maximum of 508 nm and the excitation maximum of 399 nm for our cyclic and linear forms
of GFPuv are also very close to the reported values for both wild-type
GFP (excitation maximum of 395-397 nm and emission maximum of 504 nm)
(28) and GFPuv (excitation maximum of 397 nm and emission maximum of
506 nm) (28), suggesting that there is no significant structural change. Since cyclization merely adds another loop outside the
-barrel and since linearization with thrombin converts this loop into two presumably unstructured tails, this similarity was
anticipated.

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Fig. 5.
Spectroscopic characterization of the linear
and circular forms of GFPuv. A, CD spectra of the
linear and circular forms of GFPuv. The dotted line
represent the data points of the linear form. The solid line
is the spectrum of the cyclic form. Both spectra were measured at
25 °C and pH 8.0. B, emission and excitation spectra of
the cyclic and linear forms of GFPuv. Fluorescence emission scans were
obtained by excitation at 397 nm at pH 7.5. Fluorescence excitation
spectra were recorded at an emission wavelength of 508 nm at pH 7.5. The dashed and solid lines indicate the linear
and cyclic forms, respectively. The intensities were normalized to the
peak maximum. deg, degrees.
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The secondary structures of the linear and cyclic forms of GFPuv were
compared using CD spectroscopy as shown in Fig. 5A. Both
spectra from the linear and circular forms of GFPuv show very similar
profiles, suggesting that there is no significant difference in the
secondary structure between the linear and circular forms.
Unfolding and Refolding of the Cyclic and Linear Forms of
GFPuv--
The entropically most expensive process during protein
folding is to bring the residues that are distant in the primary
sequence close to each other. Therefore, the cyclization of the protein would be expected to reduce such entropic cost during folding. In
addition, because the cyclization prevents "peeling" of terminal secondary structure elements, it might make a protein more difficult to
unfold. Hence, we were interested in elucidating if the cyclization of
GFPuv improves stability, as observed in other cyclized proteins (2,
3), and how the folding or unfolding kinetics might be affected.
However, GFP itself is already a very stable protein once its structure
has formed. The unfolding of GFPuv seems to be not fully reversible;
and due to very slow processes both in unfolding and refolding, it is
not possible to study the equilibrium precisely (18). Therefore, we
have investigated whether there is a difference in the rates of
unfolding and refolding between the circular and linear forms. These
processes were monitored by the change in the fluorescence intensity at
508 nm with an excitation of the chromophore at 397 nm. The time course
of the unfolding reactions could be fitted with a single exponential, and we assumed that the fluorescence would be zero at infinite time
(total loss of chromophore fluorescence). In Fig.
6A, unfolding of the linear
and circular forms in 6.7 M GdnHCl is followed by fluorescence intensity versus time. It is clearly visible
that the circular form unfolded more slowly than the linear form. At 7 M GdnHCl, the unfolding rate of the circular form was about half of the linear form (Fig. 6B). The unfolding rates are
plotted against the final GdnHCl concentration in Fig. 6B.
GFPuv is no more fully unfolded in 5.5 M GdnHCl (18),
preventing a comparison at low GdnHCl concentrations.

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Fig. 6.
Unfolding kinetics of the linear and cyclic
forms of GFPuv. A, typical unfolding kinetics of the
linear and circular forms of GFPuv. The measurements were performed at
pH 8.0 with a final concentration of 6.7 M GdnHCl.
B, unfolding and refolding kinetics of the linear and
circular forms of GFPuv versus GdnHCl concentrations. The
unfolding rate constants were determined by fitting the change in the
fluorescence intensity at 25 °C and pH 8 with a single exponential
(see "Experimental Procedures"). and , unfolding
rates for the linear and cyclic forms, respectively. The refolding rate
constants were obtained by fitting the change in the fluorescence
intensity with a double exponential after denaturation in 6 M GdnHCl for 3 h (see "Experimental Procedures").
The fast and slow kinetic constants are represented by
circles and triangles, respectively. The
open and closed symbols represent the linear and
circular forms, respectively. The data points were fitted by linear
regression. The dashed and solid lines represent
the linear and cyclic forms, respectively.
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In Fig. 7, we examined the refolding
process by monitoring the changes in the fluorescence intensity at 508 nm upon excitation at 397 nm. Folding was initiated by dilution in the
refolding buffer after denaturation either in 6 M GdnHCl
for 3 h (18) (Fig. 6B) or in 7 M GdnHCl for
various lengths of time. Refolding was biphasic for both molecules,
consistent with previous measurements (18), and the relative amplitude
of the slow phase was greater in linear GFP than in the circular
molecule (Fig. 7). The recovery of fluorescence appeared to be faster
for the circular form than for the linear form (Figs. 6B and
7, A and B), but this was due largely to changes
in amplitudes and not in rate constants. We found that the rate
constants for the slow phase in 0.5 M GdnHCl, refolded from
denaturation in 6 M GdnHCl, are similar for both forms
((4.0 ± 2.8) × 10
4
s
1 for the linear form and (2.6 ± 3.5) × 10
4 s
1
for the circular form) and that the time constants of the fast phase
seem to be only slightly larger for the circular form than for the
linear form ((6.5 ± 0.7) × 10
3
s
1 for the linear form and (11.4 ± 1.6) × 10
3 s
1
for the circular form). The values were the same within experimental errors whether denaturation was carried out in 6 or 7 M
GdnHCl. The rate constants did depend on final GdnHCl concentration,
however (Fig. 6B). Nevertheless, what changed dramatically
between the circular and linear forms and as a function of denaturation
time was the amplitude of both phases.

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Fig. 7.
Refolding kinetics of the linear and cyclic
forms of GFPuv. A, refolding kinetics of the linear and
circular forms of GFPuv after 3 h of denaturation in 7 M GdnHCl (pH 8.0). The reaction was started by dilution to
a final concentration of 0.5 M GdnHCl. B, same
as A, but after 20 h of denaturation in 7 M
GdnHCl. In A and B, thin and
thick lines represent the linear and circular forms,
respectively. All traces were obtained by following the intensity at
508 nm upon excitation at 397 nm. C, fraction of the fast
phase in the recovered total fluorescence versus
denaturation time. The points were fitted with an exponential function.
, circular form; , linear form.
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We consider that this is due to a slow disappearance of residual
structure in GFP, which could conceivably permit the survival of the
single cis-proline (Pro89) (15, 24, 25). The
fast phase corresponds to the folding of molecules with
Pro89 in cis, and the slow phase with
Pro89 in trans. In fact, the longer denaturation
time and higher concentration of GdnHCl reduced the amplitude of the
fast phase, probably indicating the slow loss of some residual
structure in the denatured state (Fig. 7C). The reduction of
this amplitude was clearly slower for the circular form (Fig.
7C), indicating that it maintains this residual structure
more strongly. This observation is also supported by the residual
secondary structure detected with CD spectra after denaturation in 6 and 7 M GdnHCl for 3 h (data not shown).
The analysis is complicated by the fact that longer denaturation times
dramatically reduce the recovery of the chromophore fluorescence.
Therefore, it is difficult to compare the refolding kinetics of the two
forms from an unambiguously fully denatured state. We assume that the
entropic effect of circularization, i.e. the faster initial
collapse of the chain to a productive topology, has already taken place
long before the beginning of the manual mixing measurements and would
be invisible by following the recovery of the chromophore
fluorescence. Undoubtedly, further studies will be needed to untangle
the refolding process of GFP, and the effect of circularization on any
putative stopped-flow phases or even stopped-flow burst phases will
have to be evaluated. Nevertheless, from the data presented here, there
is clear evidence that the circular molecule is kinetically protected
against chemical denaturation. This can be seen in both the rate of
loss of chromophore fluorescence (Fig. 6A) and the rate of
loss of the fast phase during refolding (Fig. 7C),
indicating loss of residual structure in the denatured state.
 |
DISCUSSION |
In this report, it is demonstrated that a cyclized protein can be
produced in vivo by making a circular permutation of a
precursor protein by artificially splitting a naturally occurring
intein domain. We used the PI-PfuI intein from P. furiosus, which has been successfully used for protein ligations
(11, 20). This permutation approach to produce a circular backbone
topology has several advantages over other in vitro as well
as in vivo cyclization approaches. Since circular
permutations of proteins have been found in nature, our experiments
indicate at least the possibility that some circular peptides might
exist that do use an intein mechanism of cyclization.
The most unique feature of our system is that mainly the cyclic form of
GFPuv was produced in the cell, as demonstrated by Western blotting,
and the pure cyclic form could be purified from the E. coli
cell extract. No by-product of the linear form was purified, which both
have often been obtained in other cyclization methods due to the
hydrolysis of the thioester bond at the C terminus of an extein before
peptide bond formation (2, 3, 9, 10). Although an unspliced precursor
remained in our system, due to much larger molecular mass, it appeared
to be mostly insoluble and thus could easily be separated from the
spliced products. Therefore, we could obtain the pure circular form
without any additional procedures to remove a by-product of the linear
form, as was necessary in our previous work (2). This separation is not
easy due to the very similar physicochemical properties of the circular
and linear forms. The absence of the linear form in the isolated
product also makes our new approach advantageous compared with a
similar in vivo cyclization approach using the naturally
split intein Ssp DnaE, which produces by-products of linear
forms even in vivo. The in vivo cyclization could
be very useful for generating intracellular cyclic peptide and protein libraries (3, 10).
We assume that the reason for the lack of contaminating linear forms is
mostly the intein used here. The products of cis-splicing with DnaE were contaminated with a product in which only the N-terminal extein piece of Ssp DnaE was cleaved off (10, 29). If the same single cleavage occurred when it was used for the cyclization reaction, this would result in a linear form. In contrast,
trans-splicing with PI-PfuI does not give rise to
single cleavage at the junction between the first extein and the intein
domain (11). This could result from a difference between
PI-PfuI and Ssp DnaE with regard to different
rates of N-S acyl migration. Nevertheless, this point requires further
investigation, as does the influence of the extein used.
Cyclized GFPuv shows some characteristic differences in molecular
properties compared with the linear form, despite the very similar
structural properties shown by CD and fluorescence spectra of both
forms. In particular, the unfolding process was clearly slower at high
concentrations of GdnHCl (Figs. 6 and 7C). These data
suggest that cyclized GFPuv is stabilized compared with the linear form
against chemical denaturation. As has been suggested previously, the
backbone cyclization could be a general strategy for stabilizing
proteins following the rationale of polymer theory (2). The detailed
mechanism of the stabilization effect is now under investigation with
other proteins in our laboratory.
In conclusion, the cyclization approach by circular permutation of the
precursor protein has been very efficient in producing a large quantity
of a circular protein, and it could be potentially useful for
generating other circular proteins. In addition, the cyclization
efficiency seems to be very high, and there are no significant amounts
of by-products of linear or polymerized forms isolated. Our in
vivo cyclization approach with its high efficiency of cyclization
may provide possibilities for several new applications of cyclic
peptides and proteins in vivo as well as for biophysical characterization of circular proteins. Selection of biologically active
cyclic proteins or peptides using a combinatorial approach with
in vivo functional selections, in vivo expression
of the cyclization-stabilized proteins without changing their primary structures, and new tools to study the biological functions of the
topology or termini in cellular environments are only some of the
interesting future studies. In particular, cyclized GFP may be
potentially useful for such studies since the unique fluorescence of
GFP has made it a convenient indicator of expression, processing, and
cellular environments.