Báo cáo Y học: Stabilization of a (ba)8-barrel protein by an engineered disulfide bridge potx

Stabilization of a (ba)
8
-barrel protein by an engineered disulfide
bridge
Andreas Ivens
1
, Olga Mayans
2
, Halina Szadkowski
3
, Catharina Ju¨ rgens
1
, Matthias Wilmanns
2
and Kasper Kirschner
3
1
Universita
¨
tzuKo
¨
ln, Institut fu
¨
r Biochemie, Ko
¨
ln, Germany;
2
EMBL c/o DESY, Hamburg, Germany;
3
Biozentrum, Universita
¨

t Basel, Basel, Switzerland
The aim of this study was to increase the stability of the
thermolabile ( ba)
8
-barrel e nzyme indoleglycerol phosphate
synthase from Escherichia coli by the introduction of disul-
fide bridges. For the de sign o f s uch v ariants, we selected two
out of 12 candidates, in which newly introduced cysteines
potentially form optimal disulfide bonds. These variants
avoid short-range connections, substitutions near catalytic
residues, and crosslinks between the new and t he three
parental cysteines. The variant linking residues 3 and 189
fastens t he N-terminus to the (ba)
8
-barrel. The rate of ther-
mal i nactivation at 50 °C of this variant with a closed
disulfide bridge is 65-fold slower than th at of the r eference
dithiol form, but only 13-fold slower than that of the
parental protein. The near-ultraviolet CD spectrum, the
reactivity of parental buried cysteines with Ellman’s reagent
as well as the decreased turnover number indicate that the
protein structure i s rigidified. T o confirm these data, we have
solved the X-ray structure to 2 .1-A
˚
resolution. The second
variant was designed to crosslink the terminal modules ba1
and ba8. However, not even the d ithiol form acquired the
native fold, possibly because one of the targeted residues is
solvent-inaccessible in the parental protein.
Keywords: indoleglycerol phosphate synthase; (b/a)

8
-barrel
proteins; stabilizing disulfide bonds; protein e ngineering.
Indoleglycerol phosphate synthase (IGPS) is a (ba)
8
-barrel
protein with an N-terminal extension of 48 residues. In
Escherichia coli, IGPS (eIGPS) is the N-terminal domain of
a monomeric, bifunctional enzyme, where the C-terminal
domain is phosphoribosyl anthranilate isomerase (ePRAI),
folded into another (ba)
8
-barrel [1]. The catalytic efficiencies
of the engineered separated domains are virtually identical
to those in the bifunctional enzyme [2]. eIGPS is, however,
more labile than ePRAI. The catalytic activity of eIGPS
decays at 55 °C w ith a half-life o f 0 .5 min [3]. In contrast,
ePRAI activity decays at 60 °C with a half-life of 100 min
(R. Sterner, Institut fu
¨
r Biochemie, Universita
¨
tzuKo
¨
ln,
Germany, personal communication). The eIGPS domain,
in turn, is also more labile than eIGPS in the native
bifunctional protein [4,5,6].
In contrast to eIGPS [1], the IGP synthases from the
hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and

Thermotoga maritima (tIGPS [3]), are thermostable, mono-
functional monomers. The comparison of the three high
resolution crystal structures suggests that an increased
number of salt bridges over that in eIGPS decreases the
rates of irreversible thermal inactivation of both sIGPS and
tIGPS. In support o f this proposal, mutational disruption of
salt bridge that crosslinks its terminal ba1andba8 modules,
significantly destabilized the variants b y comparison to the
parental enzyme [3], in support of analogous findings
reported previously [8].
The aim of this work was to stabilize the labile eIGPS
domain by introducing new disulfide bonds rather than new
salt bridges. Disulfide bonds can stabilize proteins under-
going reversible unfolding by decreasing the main chain
entropy of their unfolded states [9–11]. For example, the
most thermostable single-disulfide variants of the mono-
meric xylanase were crosslinked between the N- and
C-termini [12]. Similar observations have been made with
another monomeric (ba)
8
-barrel protein, PRAI from yeast
(yPRAI [13]). Disulfide bonds can also stabilize irreversibly
unfolding proteins by decreasing the unfolding rate [14,15].
This investigation focuses on two variants of the eIGPS
domain that are predicted to form geometrically favourable
single, new, long-range disulfide bonds, far removed from
the a ctive site and the parental cyste ines. The y either clamp
the N-terminus to the core of the (ba)
8
-barrel fold or

crosslink the ba1andba8 modules.
MATERIALS AND METHODS
DNA manipulations and sequence analysis
Preparation o f DNA samples, digestion with restriction
endonucleases, agarose gel electrophoresis, and DNA
Correspondence to A. Ivens, Universita
¨
tzuKo
¨
ln, Institut fu
¨
r
Biochemie, Otto-Fischer-Str. 12–14, D-50674 Ko
¨
ln, Germany.
Fax: +49 221 4706 731, Tel.: +49 221 470539,
E-mail:
Abbreviations: IGP, indoleglycerol phosphate; CdRP, 1-(o-carboxy-
phenylamino)-1-deoxy-
D
-ribulose-5-phosphate; PRA, N-phos-
phoribosyl anthranilate; ePRAI, PRA isomerase domain from
Escherichia coli; eIGPS, IGP synthase domain from Escherichia coli;
eIGPS-PRAI, indoleglycerol phosphate synthase–phosphoribosyl-
anthranilate isomerase bifunctional protein from Escherichia coli;
etrpC, gene encoding eIGPS; sIGPS, IGP synthase from Su lfolobus
solfataricus; tIGPS, IGP synthase from Thermotoga maritim a;Nbs
2
,
5,5¢-dithiobis(2-nitrobenzoic acid); ASA, accessible surface area.

(Received 30 August 2001, revised 30 November 2001, accepted 17
December 2001)
Eur. J. Biochem. 269, 1145–1153 (2002) Ó FEBS 2002
ligation were performed as described by Sambrook et al .
[16]. Pulsed liquid-phase sequencing was carried out on a
Applied Biosystems 477 A sequencer according to the
manufacturer’s specifications. PCRs were performed in
the Trio-block from Biometra (Go
¨
ttingen, Germany), using
thermostable Pyrococcus furiosus DNA-polymerase
(Stratagene, Heidelberg, Germany). Oligonucleotides were
purchased from Microsyn (Windisch, Switzerland).
Strains and plasmids
Protein was expressed in E. coli BL21(DE3) [F
–
ompT gal
[dcm] [Ion] hsdS
B
(r
B
–
m
B
–
)], genetic manipulation and
mutagenesis was carried out with E. coli JM109 [F¢,traD36
lacI
q
D(lacZ)M15 p roA

+
B
+
/e14
–
(McrA
–
) D(lac-proAB) thi
gyrA96(Nal
r
) endA1 hsdR17 (r
K
–
m
K
+
) relA1 supE44
recA1]. The expression vector pET21a(+), where protein
production is under control of the T7-RNA-polymerase
promoter [17], was used for expression of etrpC mutants.
Oligonucleotides
The following PCR primers were used t o amplify the etrpC
gene from the vector pMc-C/F, which contains the bifunc-
tional eIGPS:ePRAI gene [2]. The 5¢ primer was used as a
mutagenic primer for replacing Thr3 by Cys (bold letters
indicate the mutated codon). T3C 5¢ primer, 5 ¢-CGAGGG
TAA
CATATGCAATGCGTTTTAGCGAA-3¢;etrpC3¢
primer, 5¢-CCACGCGTC
AAGCTTCATACTTTATTC-3¢.

The NdeIandHindIII restriction sites for ligation into
vector pET21a(+) are underlined, respectively.
The following primers were used for replacing Arg189 by
Cys: T189C 5 ¢ primer, 5¢-AACCGTGATCTGTGCGATT
TGTCGATT-3¢; R189C 3 ¢ primer, 5¢-AATCGACAAATC
GCACAGATCACGGTT-3¢. The mutated codons are
showninboldletters.
The following four primers were used for replacing both
Ile64 and Met240 by Cys: I64C 5¢ primer, 5¢-AAAG
GCGTGTGTCGTGATGATTTCGATCCA-3¢;I64C3¢
primer, 5¢-GAAATCATCACGACACACGCCTTTTGA 3¢;
M240C 5¢ primer, 5¢-GCGTTGTGTGCCCATGACGAT
TTG-3¢; M240C 3¢ primer, 5 ¢-ATCGTCATGGGCAC
ACAACGCCGAACCAAT-3¢. The mutated codons are
shown in bold letters. The 5 ¢ prim er, which introduced
an NdeI site (underlined) into the etrpC gene for eIGPS
(64–240) was: etrpC5¢primer, 5¢-ACGAGGGTAA
CATA
TGCAAACCGTTTTAGC-3¢.
Universal T7 p romoter and terminator primers were used
for sequencing (Novagen). Both primers anneal to the T7
promoter and terminator sequences in pET21a(+), up- and
downstream of the etrpCgene.
PCR and site-directed mutagenesis
The v ector pMc2- trpC/F [2] was u sed as the template for the
production of the double mutant eIGPS(3–189) and
eIGPS(64–240). Site-directed mutagenesis was performed
by PCR with the overlap-extension method [18,19]. The
PCR mix c ontained 250 l
M

of each nucleotide triphos-
phate, 20 pmol of each primer, 0.1 lg of t emplate, 4 lLof
10 x Pfu reaction buffer and 2.5 U of Pfu -DNA polymerase
(Stratagene) in a total volume of 40 lL. The amplification
protocol for the production of megaprimers consisted of
3 min at 95 °C, followed by 35 cycles of 1 min at 95 °C,
2 min at 55 °C and 3 min at 72 °C. The megaprimers were
purified by electrophoresis on a 0.8% agarose gel. They
were used as templates in various dilutions and a t a reduced
annealing t emperature of 50 °C in a second PCR r eaction
with the 5 ¢ and 3¢ primers to yield the full-length mutated
gene. The resulting etrpC fragment was purified by electro-
phoresis on a 0 .8% agarose gel, digested with NdeIand
HindIII and purifi ed again. The fragment was then ligated
into a NdeI–HindIII digested and dephosphorylated
pET21a(+) vector, yielding the vector pET21a(+)-etrpC.
After transformation of E. coli BL21(DE3) with
pET21a(+)-etrpC, transformants w ere grown overnight in
2 m L Luria–Bertani medium [16], containing 0.1 lg amp-
icillinÆmL
)1
(Luria–Bertani/amp medium). The plasmids
were isolated and digested with NdeIandHindIII to screen
for clones with inserts. One positive clone was confirmed by
complete DNA sequencing.
Expression and purification of
E. coli
eIGPS(3–189)
The protein was expressed in E. coli BL21(DE3). Single
colonies harbouring the plasmid pET21a(+)-etrpC (3–189)

were grown overnight in Luria–Bertani medium [16],
supplemented w ith 100 lgÆmL
)1
ampicillin at 37 °C. On
the following day, 15 L Luria–Bertani/amp medium in
conical flasks were inoculated with 15 mL of the overnight
culture. The cells were allowed to grow for three days at
22 °C. After 24 and 48 h, 50 lgÆmL
)1
ampicillin was again
added. The cells were harvested a fter 64 h and suspended in
50 m
M
potassium phosphate, pH 7.8, with 1 m
M
EDTA.
For breakage of the cells, the suspension was sonified in a
Branson sonifier (2 · 2 min, level 5, 60% pulse, ice cooled).
DNase and RNase were added to a final c oncentration of
5 lgÆmL
)1
for digestion of nucleic acids. The homogenate
was centrifuged twice at 10 000 g,4°C and the supernatan t
was diluted with d eionized water to yield a conductivity of
1.27 mS Æcm
)1
.
The crude extract was loaded on a DEAE–Sepharose
fast-flow column (5 · 25 cm, 510 mL) with a flow rate of
205 m LÆh

)1
, equilibrated with 10 m
M
potassium phosphate,
pH 7.5. After washing with equilibration buffer for 1.5 col-
umn vol., the column was eluted with a linear gradient from
10 to 300 m
M
potassium phosphate buffer pH 7.5, 1 m
M
EDTA. eIGPS(3–189) eluted, as determined by activity and
SDS/PAGE, at a phosphate concentration of 150 m
M
.
Fractions containing eIGPS(3–189) were pooled and
dialyzed overnight against 5 m
M
potassium phosphate
buffer pH 6.8, 100 m
M
KCl.
The dialysate was loaded onto a hydroxylapatite column
(2.5 · 25 cm, 122 mL) with 34.2 mLÆh
)1
, that had been
equilibrated w ith 5 m
M
potassium phosphate buffer pH 6.8,
100 m
M

KCl, washedwith 1 column vol. at 68.2 mLÆh
)1
and
eluted w ith a linear gradient from 5 to 300 m
M
potassium
phosphate buffer pH 6.8, 100 m
M
KCl. At the same flow
rate, eIGPS(3–189) eluted at 1 00 m
M
potassium phosphate
pH 6.8, 100 m
M
KCl. Fractions containing eIGPS(3–189)
were pooled and concentrated by ultrafiltration to
10 mg ÆmL
)1
for a gel permeation chromatography run.
The concentrated protein solution was adjusted to a final
concentration of 300 m
M
NaCl, 3% s ucrose (v/v). The
solution was loaded on a Sephacryl S-200 column
(2.5 · 90 cm, 440 mL) equilibrate d with 50 m
M
potassium
1146 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphate buffer pH 7.5, 300 m
M

NaCl, and eluted with
equilibration buffer at a flow rate o f 34.2 mLÆh
)1
. Fractions
with pure eIGPS(3–189) were concentrated by ultrafiltration
andstoredat)70 °C after dripping into liquid nitrogen.
Enzymatic assay for indoleglycerol phosphate synthase
Indoleglycerol phosphate synthase activity was assayed at
25 °Cin50m
M
Tris/HCl pH 7.5, 1 m
M
EDTA, with 40–
70 n
M
eIGPS and 3–5 l
M
CdRP. The reaction was started
by addition of the nonfluorescent substrate C dRP [ 20].
Appearance of IGP was measured continuously by its
fluorescence excited at 280 nm a nd emitted a t 350 nm.
Because IGP accumulates, t he progress curves were fitted to
the integrated M ichaelis–Menten equation that takes com-
petitive product inhibition into account [21]. The formation
of 1 lmol IGP per minute at 25 °C was defined as one unit
of activity (Table 1).
SDS/PAGE
SDS/PAGE was carried out according to the method of
Laemmli [22]. The stacking gel and separation gel contained
6 and 12.5% acrylamide, respectively. The protein samples

were mixed with 1 vol. of 2 x SDS-sample buffer ( 100 m
M
Tris, pH 6.8, 1% SDS, 20% glycerol, 0.01% bromphenol
blue) and heated to 100 °C for 5 min before loading. The
gels were run with constant current of 30 mA for 1–2 h,
stained with Coomassie Brilliant Blue solution (0.1%
Coomassie blue, 20% acetic acid, 40% methanol) and
destained by boiling in water for 5 min in a microwave
oven. Proteins used as molecular mass standards were
bovine pancreatic trypsin inhibitor ( 6.5 kDa), myoglobin
(16.9 kDa), E. coli phosphoribosyl anthranilate isomerase
(21.1 kDa), E. coli a-tryptophan synthase (28.7 kDa),
indoleglycerol phosphate synthase (31 kDa), E. coli
b-tryptophan synthase (43 kDa), BSA (66.3 kDa) and
phosphorylase b (97.4 kDa).
Protein concentrations were determined according to
Bradford [23] with known concentrations of BSA as
standard, as well as with absorbance spectroscopy at
k ¼ 280 nm (e
eIGPS
¼ 0.81 cm
2
Æmg
)1
) in a Hewlett
Packard Diode Array spectrophotometer (model 8452 A),
connected to a HP Vectra ES/12 computer.
Protein thermal stability determined by inactivation
kinetics
For stability measuremen ts, the enzymes we re incubated in

0.1
M
potassium phosphate buffer at a given temperature
and irreversibly heat inactivated. Aliquots were taken at
certain time points and chilled on ice, until the remaining
activity was determined (in Tris, as described above) and
plotted against the incubation time. Kinetic data were
obtained a s d escribed above. Incubation buffer was 100 m
M
potassium phosphate, pH 7.5, 1 m
M
EDTA, 1 m
M
dithio-
threitol. D ithiothreitol was omitted in the case of oxidized
eIGPS(3–189).
Oxidation and reduction of the engineered disulfide
bridge in eIGPS(3–189)
For reduction of the disulfide bridge, the enzyme at a
concentration o f 1.5 mgÆmL
)1
was i ncubated f or 6 h at 4 °C
in 50 m
M
potassium phosphate pH 7.5, 300 m
M
NaCL,
10 m
M
dithiothreitol. For promoting the formation of the

disulfide bond, the enzyme was incubated overnight at 4 °C
in 50 m
M
potassium phosphate pH 7.5, 300 m
M
NaCL,
supplemented with 0 .5 m
M
Nbs
2
as the o xidizing com-
pound. To examine whether the thiols are reduced or the
disulfide bridge is formed, the protein samples were run on a
nonreducing SDS/PAGE.
Determination of thiol content
The content of free S H groups and cysteines involved in a
disulfide bridge was d etermined according t o the reaction of
Ellmann [24]. Stock solutions of assay buffer were 1 m
M
Nbs
2
in 50 m
M
Na-phosphate buffer pH 7.5, 1 m
M
EDTA
or 50 m
M
Tris pH 7.5, 1 m
M

EDTA. The following
extinction coefficients were used: e
TNB
(440 nm) ¼
9.22 m
M
)1
Æcm
)1
, eNbs
2
(325 nm) ¼ 17.38 m
M
)1
Æcm
)1
.
Excess amounts of both re ducing and oxidizing compounds
were removed before the measurements by gel filtration on
NAP columns (Pharmacia). A blank run was performed
with assay buffer before the protein was added to a final
concentration of 10–30 l
M
in a final volume of 1 mL. After
various time points the absorption at 440 nm was recorded.
CD spectra
CD spectra were monitored with a Jasco model J -720
spectropolarimeter, which was connected to a Philips SX
computer. The measurements were carried out in 0.05
M

Na-phosphate buffer, pH 7.5, 1 m
M
EDTA in the absence
of oxidizing agent for oxidized forms and in the presence o f
1m
M
dithiothreitol for reduced forms. For all CD
measurements, 10 spectra were recorded and averaged.
X-ray structure solution
Prior to crystallization, the protein buffer was exchanged in
NAP 10 columns (Pharmacia) to 50 m
M
potassium phos-
phate,pH7.5,1m
M
EDTA. The protein was then
concentrated to 10 mgÆmL
)1
in Centriprep and Centricon
ultrafiltration units (Amicon). Crystallization was carried
Table 1. Purification of the eIGPS(3–189) disulfide variant from 69 g (wet weight) of transformed E. coli cells.
Fraction
Total protein
(mg)
Total activity
(U)
Specific activity
(UÆmg
)1
)

Yield
%
Crude extract 2093 529 0.3 100
Anion exchange eluate 311 313 1.0 59
Hydroxylapatite eluate 208 208 1.0 40
Gel filtration eluate 102 239 2.3 45
Ó FEBS 2002 Stabilization of a (ba)
8
-barrel protein (Eur. J. Biochem. 269) 1147
out as reported for the wild-type monomeric eIGPS [25] in
50 m
M
potassium phosphate, pH 5 .0, 1.2
M
ammonium
sulphate and 5 m
M
EDTA.
Data were collected at the synchrotron radiation beam
line X11 (EMBL c/o DESY, Hamburg) from shock-frozen
crystals at 100 K using 30% (v/v) glycerol as cryoprotec-
tant. Data were recorded on a MAR-CCD detector in three
resolution sweeps to a maximum resolution of 2.1 A
˚
. The
crystals belong to the space group P6
3
22 and are affected by
strong pseudosymmetry. A large cell with dimensions a ¼
141.4 A

˚
2
, c ¼ 156.7 A
˚
and containing three molecules per
asymmetric unit coexists with a subcell of dimensions
a ¼ b ¼ 81.6 A
˚
2
, c ¼ 156.7 A
˚
and one molecule per
asymmetric unit. Both cells are related b y a rotation of 30°
around c. Only reflections corresponding to the subcell
(k ¼ h ±3n) show significant intensities, with reflections
from the larger cell being remarkably weaker. This crystallo -
graphic problem also affected monomeric, wild-type eIGPS
and has been described previously [25]. The structure
presented here corresponds to that of the subcell and
therefore represents an averaged model.
The
HKL
suite of programs [26] was used in data
processing and reduction. The data set consisted of 17 306
unique reflections with a multiplicity of 8.8%, an overall
R
merge
of 4.1% and a completeness of 93.7% (the outer
resolution shell, 2.15–2.10 A
˚

, had values of 25.0% R
merge
,
multiplicity 2.6% and 78% completeness). Structure solu-
tion was carried out by the molecular r eplace ment technique
(AMoRe [27]), using the eIGPS domain from the bifunc-
tional enzyme [1] as a search model. For refinement,
reflection data were divided into a working set and a t est set
(1057 reflections) u sing
FREERFLAG
. Refinement was carried
out using the
CNS
software [28] and included bulk solvent
correction, overall anisotropic B-factor scaling and
restrained, individual, isotropic B factor refinement. The
structure has been refined to a crystallographic R-factor of
24.1% (R
free
31.9%). The model includes protein residues
1–259, the CdRP compound and 187 solvent atoms. No
obvious interpretable electron d ensity can b e observed for
residues 1 and 2, so these were included as model s.
The coordinates and structure factors have been depo-
sited at the PDB with accession code 1218477 (1JCM).
RESULTS AND DISCUSSION
Design of disulfide bonds
Engineering of a new disulfide bond into eIGPS must take
into account the presence of three parental cysteines within
the s trands b

1
(C54), b
3
(C113) and b
4
(C134) o f the ( ba)
8
barrel (Fig. 1). At first sight, any of these native cysteines
might be used as disulfide-bonding partners. All three
residues are, however, solvent-inaccessible: the A SA values,
calculated with AREAIMOL [28a], are 1, 2 , and 0 A
2
,
respectively. Although they are not conserved [1], and
therefore n ot directly essential f or catalysis, C54 and C113
are nevertheless adjacent to two catalytically essential
residues, namely E53 and K114 [5]. Initially, 12 potential
residue pairs with an appropriate geometry for disulfide
formation were identified, using the
MODIP
program [29].
Ten of these pairs were rejected, however, using the
following criteria for exclusion: (a) pairs of residues le ading
to short-ran ge d isulfides, that is, separated by less than 25
positions in the s equence; (b) positions adjacent to catalytic
residues; a nd (c) new cysteines leading to geometrically
favourable disulfides [29] with one of the three parental
cysteines [30]. We avoided using the parental cysteines as
partners for new disulfides, because the orientation of E53
and K114 might be altered by the introduction of an

adjacent disulfide bridge, thus impairing catalysis.
One of the preferred, new disulfide bonds requires the
double substitution T3C/R189C, and fi xes t he N-terminus
to the barrel core of this variant, designated eIGPS(3–189).
The disulfide bond is accessible (ASA values of T3 and
R189 are 54 and 68 A
˚
2
, respectively) and fortuitously
mimics one o f the e xtra salt bridges in both sIGPS [7] and
tIGPS [3], which are missing in eIGPS [1]. However, the
replacement R189C in eIGPS disrupts the parental short-
range salt bridge of R189 to E169 on helix a5
.
The other selected disulfide variant involves the double
substitution I64C/M240C (ASA values o f I 64 and M240 are
27 and 0 A
˚
2
, respectively), and is de signated eIGPS(64–
240). M240 is an invariant but solvent-inaccessible residue
that anchors t he short h elix a8¢ to the core o f the protein [1].
This proposed disulfide crosslinks the loops b1a1andb8a8,
which are widely separated i n sequence but adjacent i n space
(Fig. 1 ), thus clamping the barrel between the N- and
C-terminal modules ba
1
and ba
8
. This disulfide bond is

topologically analogous to the strongly stabilizing disulfide
bond introduced between helices a1anda8ofthemono-
meric (ba)
8
-barrel protein yPRAI from yeast [13], and
fortuitously mimics the s tabilizing salt bridge E73-R241 in
tIGPS [3].
Production and purification of disulfide-bonded proteins
The eIGPS variants ( 3–189) a nd (64–240) were produced by
growing transformants of E. coli strain BL21 (DE3), as
Fig. 1. Stereo representation of i ndoleglycerol
phosphate synthase from E. coli. The bound
phosphate ion indicates the location of the
active site. The Ca positions of native cysteines
(54, 113 a nd 134) and of the p lanned d isulfide
bonds (3–189) and (64–240) are shown. 15,
position of the single t ryptophan residue.
1148 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
described in Materials and methods. As generally a larger
fraction of soluble protein is expressed during c ell growth at
temperature lower than at 37 °C [31], the cells were grown at
22 °C for 64 h. As estimated by SDS/PAGE, 30% of the
variant (3–189) remained so luble under these c onditions. It
was purified from the soluble fraction of the cell homogen-
ate in the absence o f dithiothreitol, to promote spontaneous
formation of the disulfide bond by auto-oxidation. Chro-
matography, fi rst o n an anion exchange r esin, then on
hydroxylapatite and finally on a size-exclusion gel, resulted
in preparations that were at le ast 9 5% pure, as e stimated by
SDS/PAGE under r educing conditions (in the presence of

2-mercaptoethanol; Fig. 2B), and with an overall yield of
45% (Table 1).
When the (3–18 9) protein was analyzed by SD S/PAGE
under nonreducing conditions, two bands of about equal
strength were observed (Fig. 2A). Because the spontaneous
formation of the disulfide bond was apparently incomplete,
total oxidation was achieved by incubating the protein with
an excess of Ellman’s reagent (Nbs
2
[32]), leading to a single,
but faste r migrating band (Fig. 2C). SDS micelles decorated
with disulfide-bonded proteins have a smaller hydrodynamic
volume than those d ecorated with the corresponding dithiol
forms [33], and therefore migrate more rapidly.
In contrast, during culture at 22 °C of the cells producing
the ( 64–240) va riant, most of the protein partitioned into the
insoluble fraction of the cell homogenate. Attempts to
purify this variant by first solubilizing the pre cipitate in
guanidinium chloride in the presence or absence of
dithiothreitol, an d then dialyzing a gainst phosphate buffer
[5], failed to yield significant amounts of soluble material.
Apparently, parallel substitution of residues 6 4 and 240 by
cysteines prevents the correct folding of the enzyme. The
available IGPS structures [1,7] suggest t hat replacement of
the long and hydrophobic side chain of the buried and
invariable residue M240 by the short, polar side-chain of
cysteine m ay disrupt the abundant hydrophobic i nteractions
at the C-terminus and hinder the correct folding of the
protein. Stu dies with this variant w ere therefore not pursued
further.

Crystal structure of the oxidized variant eIGPS(3–189)
In order to assess the extent to w hich the ( 3–189) disulfide
bond had actually formed in the partially oxidized variant
eIGPS(3–189) (see Figure 2A), the crystal structure of this
protein was solved by X-ray crystallography to 2.1-A
˚
resolution. Unfortunately, t he 4-A
˚
resolution of the previ-
ous crystal structure of the monofu nctional e IGPS [25]
obstructs comparison to both the structure of the eIGPS
domain of the bifunctional enzyme and the structure of the
variant reported here. Hence, throughout this work, the
structure of the eIGPS domain from the bifunctional
enzyme [1] will be u sed as r eference. The Ca tr ace of
partially oxidized eIGPS(3–198) very closely superimposes
on that of the e IGPS domain (the overall rmsd for Ca atoms
is 0.51 A
˚
), with residues 45–47 and 206–209 involved in
crystal packing showing the largest differences.
Figure 3 s hows t he enzyme in a state of partial oxidation,
as confirmed by SDS/PAGE from dissolved crystals
(Fig. 2 A). Indeed, double conformations can be observed
for the side-chain of the C3 residue, with one rotamer as
part of the d isulfide bridge to C189, and a second rotamer
corresponding to the -CH
2
SH side chain of the reduced
form. Overall, the structure does not reveal any substantial

differences compared to the wild-type eIGPS in the vicinity
of the substitutions. Additional electron density was
observed, however, in the active site of the protein. It can
be modelled as the CdRP substrate (data not shown), but a
detailed description including a comparison to related
Fig. 2. Partial oxidative closure of the engineered (3–189) disulfide
bond. SD S PAGE in a bsence of mercaptoethanol. Lane A, purified
and spontaneously oxidized, variant (3–189); l ane B, as in (A), but with
dithiothreitol in the sample buffer; lane C, as in (A), but with Nbs
2
;
lanes M, marker proteins with the given M
r
-values (kDa).
Fig. 3. 2F
obs
-F
calc
/a
calc
electron density map
showing the disulfide bond contoured at 1.0 r.
The newly introduced cysteine residues C3 and
C189 are l abelled (black dots, Sulfur atoms).
Ó FEBS 2002 Stabilization of a (ba)
8
-barrel protein (Eur. J. Biochem. 269) 1149
complex structures will be reported elsewhere. Perhaps this
unexpected feature is responsible for the observed incom-
plete autoxidation of eIGPS(3–189) shown in Fig. 2A. The

following measurements were conducted with the completely
oxidized form (Fig. 2 C).
Conformational analysis in solution
In order to further analyze possible structural differences
between the reduced and o xidized f orms of eIGPS(3–189),
designated red(3–189) and ox(3–189), a nd the wild-type
eIGPS, we measured far-UV CD spectra in phosphate
buffer (Fig. 4). All forms displayed identical spectra within
error limits. Therefore, it can be concluded that secondary
structural elements are not perturbed by the double
replacement in both the d ithiol a nd disulfide forms. Near-
UV CD measurements were performed t o further analyze
the tertiary structure of eIGPS and its variants. The spectra
(Fig. 5) r evealed that eIGPS, red(3 –189) and ox(3–189)
have the same minima and maxima, indicating that they
have a similar chiral environment for W 15, which is the only
tryptophan of the eIGPS domain, and partially accessible to
solvent (ASA ¼ 48 A
˚
2
). Furthermore, fluorescence mea-
surements in phosphate buffer, excited at 295 nm, were
employed to monitor polarity changes in the environment of
W15 upon disulfide formation (data not shown). The
spectra of eIGPS, re d(3–189) and ox(3–189) were identical,
implying that the indole moiety of W15 is similarly exposed
to solvent in all three cases. The spectra were also
characterized by identical fluorescence emission maxima at
348 nm, supporting the conclusion that the indole moiety of
W15 is exposed to solvent. Thus, no significant structural

differences local to helix a0 s eem to occur in both red(3–189)
and o x(3–189), w ith respect to the wild-type. In summary,
our near-UV and far-UV CD as well as fluorescence
measurements confirm that n either the introduction of the
(3–189) disulfide bridge nor specific experimental c onditions
affect the structure of the eIGPS dom ain.
Thermostability
eIGPS can be reversibly unfolded by GdmCl in both Tris
[34] and phosphate [35] buffers. Red(3–189) displays the
same properties (data not shown). However, the unfolding
of ox(3–189) by GdmCl in the absence of dithiothreitol was
irreversible, presumably due to thiol-disulfide scrambling
[30]. Therefore, t he relative stability of the three forms could
only be estimated by irreversible thermal inac tivation
(Fig. 6 ) [14]. The results show that the maximal velocities
(V
max
¼ k
cat
· [E
0
]) of the three forms decay irreve rsibly
and exponentially at 50 °C. In contrast to eIGPS, ox(3–189)
is stabilized 13-fold, whereas red(3–189) is destabilized
fivefold, most likely due to the loss of the salt bridge E167-
R189. In other words, ox(3–189) is stabilized 65-fold over
the d ithiol form, which is the correct reference for estimating
Fig. 4. Far-UV CD spectra at 25 °C. s,parentaleIGPS;h, reduced
(dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189).
Protein concentrations were between 10 and 21 l

M
;d ¼ 0.1 cm.
Buffer: 0,05
M
Na-phosphate, pH 7.5, 1 m
M
EDTA (in case o f red(3–
189) with 1 m
M
dithiothreitol).
Fig. 5. Near-UV CD spectra a t 25 °C. s,parentaleIGPS;h, r ed uced
(dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189).
Protein concentrations were between 10 and 21 l
M
;d ¼ 5 cm. Buffer
as in Fig. 4.
Fig. 6. The disulfide bond of ox(3–189) stabilizes the fold of eIGPS
kinetically. Therm al inactivation at 50 °C i s an irreversible, exponen-
tial process. eIGPS, reduced (dithiol) and oxidized (disulfide) variants
of eIGPS(3–189) were in cubated at concentrations of 10 l
M
protein
in 0.1
M
potassium phosphate pH 7.5, 1 m
M
EDTA. eIGPS and
red(3–189) also c ontained 1 m
M
dithiothreitol. Enzyme activity was

determined in samples drawn at the indicated times and quenched on
ice. Half-lives: ox(3–189), 49 min; eIGPS, 3.7 min; red(3–189),
0.75 min .
1150 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the effect of closing this disulfide bridge. These results imply
that the engineered disulfide bond crosslinks parts of the
structure that p robably separate in t he parental protein
before the rate-determining step of i ts irreversible unfolding
is attained [15]. Thus, t he d isulfide-linked variant apparently
unfolds via a transition state that is different from t o that of
the wild-type eIGPS. Variation of the phosphate concen-
tration between 5 a nd 100 m
M
revealed that the kinetic
stabilities of eIGPS and its variants increase with i ncreasing
phosphate concentration (data not shown). These observa-
tions support t he idea that phosphate may serve as an
additionally stabilizing electrostatic clamp within the active
site. Note that phosphate specifically interacts with K55
(loop b1a1), G216 ( loo p b7a7) (not shown i n Fig. 1) a nd the
helix dipole of helix a8¢ [1], i.e. protein segments that are far
apart in the protein sequence but adjacent in s pace.
These considerations could also explain why the second
disulfide variant (64–240) does not fold properly, even in
the presence of phosphate. M240 is an invariant, solvent-
inaccessible residue (ASA ¼ 0A
˚
2
) that anchors t he short
helix a8¢ to the core of th e protein [1]. Merz et al. [36] have

shown that disrup tive substitutions near the closure
between the ba1andba8 modules of the (ba)
8
-barrel of
sIGPS are generally destabilizing. Replacing M240 of
eIGPS with cysteine may therefore destabilize the hydro-
phobic interface between the terminal modules ba1and
ba8, as well as helix a8¢, t hus decreasing the protein’s
affinity for ph osphate.
Reactivity of cysteines as a measure for protein flexibility
The accessibilities of the three buried cysteine s (C54, C113
and C134) of eIGPS and the dithiol and disulfide forms of
the (3–189) variant can be assessed by measuring the
kinetics of their irreversible reaction with Nbs
2
[32] [37].
ESH þ DTNB À!
k
ESTNB þ TNB ð1Þ
This reaction, which can be followed by the increase of
absorption of TNB at 440 nm, becomes pseudo-first order,
i.e. exponential, when Nbs
2
is in excess:
Àd½ESH
dt
¼
d½TNB
dt
¼ k

obs
½ESð2Þ
where k
obs
¼ k[DTNB] is the observed first-order rate
constant. Both the total number of reactive cysteine
sulfhydryls as well as their average rate constant k as
expressed b y t he observed half-lives t
1=2
¼
ln 2
k½DTNB
are
presented in Table 2. The measurements were performed
in 50 m
M
phosphate buffer, when the active site is 97%
saturated with phosphate [38].
As determined by SDS/PAGE under nonreducing condi-
tions (as described in Fig. 2), no i ntermolecular bridges were
formed during the oxidation process, as there was no
evidence for e ither aggregation o r cross-linking. The possi-
bility t hat a further i ntramole cular disulfide bridge had
formed between C113 and C134 (cf. Fig. 1) was also
excluded b y measurements of the forms in 0.5% SDS, which
unfolds the proteins immediately and allows Nbs
2
to react
with all free thiols that were not accessible in the folded
state. No decrease of the maximally expected numb er o f free

sulfhydryl groups was found.
Only one of three native cysteines of eIGPS reacted with
Nbs
2
, a lbeit slowly. A s the three cysteines are basically
solvent inac cessible, as judge d from the calculated a ccessible
surface areas (ASA values are: C45 ¼ 1A
˚
2
;C113¼2A
˚
2
;
C134 ¼ 0A
˚
2
), we cannot identify the reactive cysteine of
eIGPS from structural considerations. In ox(3–189), how-
ever, in which the two newly introduced cysteines form a
disulfide bond, the reactive cysteine is almost completely
protected.
In contrast, at least two cysteines of red(3–189) react
rapidly. As the positions of T3 and R189 of eIGPS are
partially accessible to solvent (ASA values are: T3 ¼ 54 A
˚
2
;
R189 ¼ 68 A
˚
2

), it is likely that C3 and C189 of red(3–189)
are the two reactive cysteines. They are converted to the
corresponding disulfide via a mixed disulfide intermediate
[E(SH)STNB], which does not accumulate.
EðSHÞ
2
þ DTNB ! EðSHÞSTNB þ TNB ð3Þ
EðSHÞSTNB ! ES
2
þ TNB ð4Þ
The cysteine group in excess of C 3 and C189 that reacts
to 40% c ompletion in r ed(3–189) is likely the same as that
which reacts in eIGPS to 90%. The particularly slow
reaction of this parental cysteine in ox(3–198) to only 10%
completion must be due to the decreased structural
fluctuations of this form of the variant hindering the access
of DTNB by comparison to eIGPS.
Catalytic constants
As the active s ite is l ocated in a d epression at the C-terminal
end of the b-barrel, between the structured segments that
carry the newly introduced pairs of cysteines (see Fig. 1),
enzyme activity is a sensitive monitor for detecting changes
in both the structure and flexibility of the three enzymes.
Steady-state kinetic measurements were conducted in Tris
Table 2. Reaction of protein sulfhydryl groups with Nbs
2
. The protecting effect of th e introduced disulfide bridge.
Variants
Free sulfhydryl groups per protein chain
a

Total
b
Accessible Protected
c
t
d
1=2
(min)
eIGPS 3 0.9 2 10
ox(3–189) 3 0.1 3 > 100
red(3–189) 5 2.4 3 < 1
a
Buffer: 0.05
M
potassium phosphate buffer, pH 7.5, 1 m
M
EDTA; T ¼ 25 °C.
b
Evaluated by conducting the reaction at 25 °C in 0.05
M
Tris buffer, pH 7.5, 0.5% SDS.
c
Rounded, integral numbers.
d
Half-life evaluated from exponential progress curves recorded at 440 nm.
Nbs
2
concentration ¼ 1m
M
.

ÓFEBS 2002 Stabilization of a (ba)
8
-barrel protein (Eur. J. Biochem. 269) 1151
buffer and i n the absence of dith iothreitol. Measurements in
phosphate buffer are not feasible because phosphate is a
competitive inhibitor (K
i
¼ 2.8 m
M
[38]). The Michaelis
constants (K
CdRP
M
) of the two forms of both ox(3–189) and
red(3–189) are only % 15% smaller than that of eIGPS
(Table 3). The turnover numbers, however, are decreased t o
45% in red(3–189) and to 10% in ox(3–189). A s t he poor
activity of the thermostable IGPS from S. solfataric us at
low temperature is due to the rate-limiting release of the
product IGP [36], it i s likely that ox(3–189) is ‘constipated’
[36] by the rigidified structure. This finding suggests that
covalent crosslinking the helix a0 to the loop b6a6is
responsible for the retarded release of product in ox(3–189),
and i s supported by the decreased reactivity with Nbs
2
of the
single most reactive cysteine in o x(3–189), in contrast to
eIGPS (Table 2).
CONCLUSION
We have demonstrated that a mesophilic (b/a)

8
-barrel
enzyme from the tryptophan biosynthesis pathway, namely
indoleglycerol phosphate synthase from E. coli,canbe
stabilized against irreversible thermal denaturation by the
introduction of a new disulfide bridge. The new disulfide
crosslink of eIGPS(3–189) fastens the N-terminal extension
to the catalytic face of the (ba)
8
-barrel fold, thus rigidifying
it and changing the pathway of unfolding. Despite obeying
the structural criteria of good disulfide geo metry, as well as
sufficient distance from both catalytic residues and parental
cysteines, the variant eIGPS(64–240) failed to fold to the
native structure, even in the r educed state. We conclude that
another important criterion is to avoid replacing solvent-
inaccessible, hydrophobic residues by cysteines. Although
surface disulfide bridges have not generally been selected
during the evolution of thermophilic proteins [39], p erhaps
because of the chemically reducing e nvironment of the
cytoplasm, correctly d esigned disulfide bridges, which form
only after release from the cytoplasm, are o f specific interes t
for biotechnological applications.
ACKNOWLEDGEMENTS
The authors thank Drs Thorsten Kno
¨
chel and Ralf Thoma for advice,
Dr Reinhard Sterner for critical discussion and Gu
¨
nter Pappen berger

for designing the stereo figure. This work was supported by grant Nr.
31–45855.95 of the Swiss National Science Foundation (to K. K.).
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