Tài liệu Báo cáo khoa học: Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding pdf

Local stability identification and the role of a key aromatic
amino acid residue in staphylococcal nuclease refolding
Zhengding Su
3
, Jiun-Ming Wu
1
, Huey-Jen Fang
1
, Tian-Yow Tsong
2,3
and Hueih-Min Chen
1
1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC
2 Institute of Physics, Academia Sinica, Taipei, Taiwan, ROC
3 Department of Biochemistry, University of Minnesota College of Biological Sciences, St Paul, MN, USA
Staphylococcal nuclease (SNase) is a single-domain
protein of 149 amino acids. Its 3D structure has been
examined by NMR [1–3] and X-ray crystallography
[4,5]. However, only part of the structure (positions 1–
141) can be confirmed. The segment 142–149 has not
been defined with certainty because of its apparent
flexibility. Structural observations made with NMR or
X-ray have led to the prediction that certain amino
acid(s) in the flexible segment stabilize the rest of the
structure, in particular the key amino acid(s) located
close to this flexible segment. The tryptophan at posi-
tion 140, for example, may play an important role in
maintaining the protein structure formed by amino
acids 1–141. In this study, we used site-directed muta-
genesis to generate point mutations and truncations
around this position to explore the above prediction.

As shown by Chen and colleagues [6], SNase protein
can be unfolded by lowering its pH (for example, from
pH 7 to pH 2). About 2.5 protons are associated with
the key glutamic amino acid residues at positions 75 and
129. This association between protons and key amino
acids leads the protein to unfold. However, the refolding
process may be different and more complex because the
transition of refolding is from many unfolded states to
a single native state. Based on previous kinetic experi-
ments using single-jump and double-jump stopped-flow
for refolding [7,8], SNase protein can be refolded in vitro
to its active 3D conformation in milliseconds. The
sequence of equilibrium reactions between the three
denatured states and one native state can be shown as
[9] N « D
1
« D
2
« D
3
, where N is the protein in its
native state and D
i
(i ¼ 1–3) indicates the protein in its
unfolded state. This scheme has been used to solve puz-
zles such as accumulated intermediates and to conduct
random searches among ‘microscopic states’ [8]. The
early stages of refolding (D
i
fi N) occur via key amino

acid(s) which act as nucleation centres before proton
dissociation. Subsequently, these centres trigger the
condensation of random polypeptide chains into the
compact form of the native state.
In this study, the effects of mutating W140 [10]
on SNase protein conformation and stability were
Keywords
aromatic amino acid; refolding; stability;
staphylococcal nuclease
Correspondence
H-M. Chen, Institute of BioAgricultural
Sciences, Academia Sinica, Taipei,
Taiwan 115, R.O.C.
Fax: +886 2 2788 8401
Tel: +886 2 2785 5696 ext. 8030
E-mail:
(Received 3 May 2005, revised 3 June
2005, accepted 13 June 2005)
doi:10.1111/j.1742-4658.2005.04814.x
Staphylococcal nuclease (SNase) is a model protein that contains one
domain and no disulfide bonds. Its stability in the native state may be
maintained mainly by key amino acids. In this study, two point-mutated
proteins each with a single base substitution [alanine for tryptophan
(W140A) and alanine for lysine (K133A)] and two truncated fragment
proteins {positions 1–139 [SNase(1–139) or W140O] and positions 1–141
[SNase(1–141) or E142O]} were generated. Differential scanning micro-
calorimetry in thermal denaturation experiments showed that K133A and
E142O have nearly unchanged DH
cal
relative to the wild-type, whereas

W140A and W140O display zero enthalpy change (DH
cal
 0). Far-UV CD
measurements indicate secondary structure in W140A but not W140O, and
near-UV CD measurements indicate no tertiary structure in either W140
mutant. These observations indicate an unusually large contribution of
W140 to the stability and structural integrity of SNase.
Abbreviations
DSC, differential scanning calorimetry; SNase, staphylococcal nuclease.
3960 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS
investigated. Two point-mutated proteins, with a single
base substitution of alanine for tryptophan (W140A)
and alanine for lysine (K133A), and two truncated
fragment proteins, with fragments 1–139 [SNase(1–
139) or W140O] and 1–141 [SNase(1–141) or E142O],
were generated. The effect of these C-terminal trunca-
tions and point mutations to C-terminal residues on
SNase stability and conformation integrity was exam-
ined by subjecting these mutants and wild-type pro-
teins to CD [11,12] and differential scanning
calorimetry (DSC) [13,14]. The preliminary refolding
process in terms of assembly of fragments is discussed.
Results
CD spectra
Secondary structure of SNase and its mutants was deter-
mined by CD with spectrapolorimetry measurements.
The approximate fraction of secondary structure type
that is present in any protein can be determined by its
far-UV CD spectrum as the sum of fractional multiples
of the reference spectra for each structural type.

Figure 1A shows the CD spectra of W140A, E142O
and K133A. There are no significant differences from
that of the wild-type protein. In contrast, W140O has a
completely different CD spectrum. There are no peak
minima at 222 nm and 208 nm, but instead a peak mini-
mum at 200 nm, indicating that it has no secondary
structure only random coil. We can therefore postulate
that, when the segment beyond W140 was removed, the
secondary structure of the protein disintegrated. Using
the peak at 222 nm of the wild-type protein as an index
of helical conformational stability, this CD spectrum of
W140O suggests a decrease in helicity on removal of all
amino acids beyond position 140.
In CD spectra in the near-UV region (Fig. 1B), the
wild-type protein and mutants E142O and K133A show
strong intensity at  277 nm (h  )77 degreesÆcm
)2
Æ
dmol
)1
), revealing an intact tertiary conformation. In
contrast, W140A and W140O both lacked tertiary struc-
ture, as their intensities at 277 nm were only h  )22
and )13 degreesÆcm
)2
Ædmol
)1
(Fig. 1B). However,
W140A at 295 nm had a similar spectrum to that of the
wild-type protein, but W140O did not (h  0). This may

indicate that the aromatic F or Y in the W140A mutant
are more ordered and compact than in the W140O
protein.
Tryptophan fluorescence spectra
W140 is located near the flexible C-terminus of SNase.
Changes in the fluorescence intensity of W140 reflect
a change in the hydrophobic environment surrounding
W140 and thus indicate a change in the overall (ter-
tiary) structure of the protein. Figure 2 shows the
fluorescence spectra of wild-type SNase and the four
mutants E142O, K133A, W140A and W140O. The
fluorescence spectra of E142O and K133A are similar
to that of the wild-type protein. However, the fluores-
cence in the mutants without tryptophan at 140
(W140A and W140O) was much lower than both the
wild-type protein and mutants E142O and K133A.
Thermal analysis of protein unfolding
The DSC curves of the wild-type protein and the
mutants K133A, E142O, W140A and W140O are
shown in Fig. 3, with their thermodynamic parameters
summarized in Table 1. The calorimetric DH
cal
values
Fig. 1. CD spectra of wild- type and SNase mutants. (A) CD spectra
(far-UV) of five proteins [wild-type (WT), W140A, E140O, W140O
and K133A]. All spectra are similar except the W140O spectrum
(bold line). (B) CD spectra (near-UV) of the same proteins. Protein
concentration was 0.5 mgÆmL
)1
.

Z. Su et al. Staphylococcal nuclease refolding
FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS 3961
of the wild-type protein and K133A and E142O were
88.45, 84.70, and 83.60 kcalÆmol
)1
, respectively. The
melting point and DSC profile of E142O (52.8 °C) was
almost identical with that of the wild-type protein
(52.32 °C). K133A had a similar DH
cal
but a slightly
different melting point (47.70 °C) compared with the
wild-type protein. These results suggest that lysine at
position 133 and the residues that follow glutamic acid
at position 142 play negligible roles in maintaining the
native structure of SNase.
In contrast, W140A and W140O revealed no distinct
maxima in their DSC profiles. This is very different
from the wild-type protein and K133A and E142O,
which showed a significant enthalpy change. This
implies that either the protein has poor stability or no
secondary ⁄ tertiary structure results from either repla-
cing the tryptophan at position 140 with alanine or the
removal of a fragment from positions 140–149. This
is supported by the melting points of the proteins
(52.32 °C for the wild-type and ‘not detectable’ for
W140A and W140O). Flanagan et al. [15] reported
that multiple mutations can cause large changes in the
average conformation of denatured proteins. Here we
show that a specific single mutation or removal of a

specific fragment can cause large changes in the native
state of SNase.
Discussion
In addition to its local electrostatic interactions, which
contribute largely to SNase stability via specific
charged amino acids (Results shown in the preceding
paper), other nonelectrostatic interactions at even more
specific positions play a significant role in maintaining
SNase tertiary structure.
Our CD and DSC data show that the W140 in
SNase is the amino acid responsible for the stability of
the whole protein. However, in comparison with the
wild-type protein, the mutant W140A retains signifi-
cant secondary structure (Fig. 1A). Yin & Jing [16]
reported that W140 plays an important role in the
native-like SNase conformation and the enrichment
of an ordered secondary structure. If tryptophan is
relocated from position 140 to 34, the mutant
(F34W ⁄ W140F) [17] retains its secondary structure,
but its tertiary structure is lost. This implies that tryp-
tophan plays a significant role in maintaining the 3D
structure only at 140 (wild-type SNase has only one
W). Without W140, the secondary structure may be
maintained by other interactions such as local stable
segments interacting around E75 and E129, areas with
oppositely charged amino acids. The truncated protein
W140O in this study (deletion of residues 1–140) shows
zero enthalpy on thermal unfolding (Table 1) and has
no secondary structure (Fig. 1A). This indicates that,
without tryptophan and residues 140–149, the protein

Fig. 2. Steady-state fluorescent spectra of wild-type and mutant
SNase. Spectra of five proteins [wild-type (WT), W140A, E140O,
W140O and K133A]. Protein concentration was 0.4 mgÆmL
)1
.
Fig. 3. Calometric melting curves of wild-type and mutants of
SNase. DSC curves of five proteins [wild-type (WT), W140A,
E140O, W140O and K133A]. The curves of W140A and W140O
are nearly linear in terms of intensity. All protein concentrations
were 2 mgÆmL
)1
.
Staphylococcal nuclease refolding Z. Su et al.
3962 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS
cannot maintain its tertiary conformation or even the
secondary motif. However, if residues 142–149 are
removed, the protein largely retains its 3D structure
(Table 1 and Fig. 2). Related experiments have been
performed previously. Parker et al. [18] showed that
the apparent association constant of SNase(1–126)
bound to pdTp in the presence of Ca
2+
is approxi-
mately threefold lower than that of the wild-type pro-
tein. Therefore, some or all of the residues in the 127–
149 segment are critical for maintenance of the native
conformation of the protein. In addition, Griko and
coworkers [19], studying SNase(1–136), showed that
this fragment (residues 1–136) has no secondary struc-
ture but retains the tertiary conformation. They thus

proved that SNase(1–136) is not a ‘molten globule’
protein, although its unfolding process was a first-
order phase transition. This situation contrasts with
our W140O [SNase(1–139)] mutant, which maintains
its secondary structure to a certain extent but has no
tertirary conformation (Fig. 1A).
As to the point of fragment assembly during protein
folding, Anfinsen and coworkers [20,21] originally
studied the enzymic effect using overlapping structure-
less fragments [for instance, SNase(99–149) interacting
with SNase(1–126)] and found that the reconstructed
complex was active and exhibited much of the ordered
structure. Anfinsen consequently predicted that this
protein was organized from its polypeptide chain into
N-terminal and C-terminal centers during refolding
[21]. Since then, many scientists have been interested in
demonstrating this concept by using N-terminal and
C-terminal fragments of SNase as models for the study
of folding and unfolding pathways. For example, Choy
& Kay [22] using NMR
15
N and
2
H spin relaxation
techniques illustrated that the fragment D131D (dele-
tion of wild-type residues 3–12 and 141–149) of SNase
was completely unfolded under nondenaturing condi-
tions (pH  5 and low salt concentration). This is
because they observed much more vibrational motion
in the unfolded protein than in folded ones on the

picosecond time scale. Taniuchi et al. [23] used both
SNase(1–126) and SNase(50–149) as typical fragments
to form type I and type II (in equilibrium) complex
proteins. They reported that these two overlapping
fragments could form enzymically active complement-
ary structures, and their folding rates were not related
to any decrease in energy from the unfolded to the
folded state. Feng et al. [24] created the SNase frag-
ments SNase(1–110), SNase(1–121) and SNase(1–135)
and used them in NMR spectroscopy experiments to
study the folding process. They concluded that the
conformation of these fragments could be considered
as native-like partially folded and unfolded states.
Recently, they further used the short N-terminal frag-
ments SNase(1–20), SNase(1–28) and SNase(1–36) to
show that the folding nucleation sites of SNase may
start from the N-terminus [25]. However, we used the
C-terminal fragments SNase(1–140) and SNase(1–142)
to show that SNase(1–140) plays a role in the assembly
of the protein during refolding. Our results also show
that SNase(1–140), without Trp at position 140, does
not have any structure, whereas the fragment including
Trp at position 140, i.e. SNase(1–142), has a similar
structure to the wild-type enzyme.
Our experiments show that tryptophan at position
140 plays an important role in maintaining protein ter-
tiary integrity. Figure 4A shows how tryptophan (loop
1) may interact with loop 2 and loop 3. These inter-
actions seem to form a ‘lower neck’ in the protein as
compared with the ‘upper neck’ which is formed by

E75 with H121 and K915. The forces responsible for
the interactions between these loops are not yet clear.
One possibility is that the loops together with W140
constitute a nucleation center. The indole ring [26]
of W140 may be the main target for the interaction
with both loop 2 and loop 3. Furthermore, from the
protein folding scheme discussed previously [9],
N « D
1
« D
2
« D
3
, the energy needed for transfor-
mation among the D
i
states of protein unfolding ⁄
refolding is negligible (4.57 kcalÆmol
)1
for D
1
« D
2
and 4.32 kcalÆmol
)1
for D
2
« D
3
) and a possible

intermediate state such as SNase(1–139) exists
Table 1. DSC results of SNase and its mutants. Phosphate buffer (25 mM Na
2
HPO
4
⁄ 50 mM NaH
2
PO
4
⁄ 200 mM NaCl, pH adjusted to
7.0) was used in the experiments. All proteins were used at a concentration of 2 mgÆmL
)1
. Difference from DH of WT (%) calculated by
[(DH
mutant
) DH
WT
) ⁄DH
WT
] · 100. Difference from DC
p
of WT (%) calculated by [(DC
p mutant
) DC
pWT
) ⁄DC
pWT
] · 100. WT, Wild-type; ND,
not detectable.
Average T

m
(°C) DH (kcalÆmol
)1
)
Difference of
DH from WT (%)
DC
p
(kcalÆmol
)1
ÆK
)1
)
DDC
p
(kcalÆmol
)1
ÆK
)1
)
Difference of
DC
p
from WT (%)
Wild-type 52.32 ± 2.5 88.45 ± 1.40 2.40 ± 0.20
K133A 47.70 ± 0.35 84.70 ± 2.5 )1.40% 2.38 ± 0.05 )0.02 )0.8%
E142O 52.80 ± 0.55 83.60 ± 3.5 )2.68% 2.41 ± 0.04 0.01 0.4%
W140A ND ND )100% ND ND )100%
W140O ND ND )100% ND ND )100%
Z. Su et al. Staphylococcal nuclease refolding

FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS 3963
(Fig. 4B, State 2). When tryptophan occupies position
140, the whole protein is folded into its native confor-
mation (Fig. 4B, State 3).
Summary
Trp140 located in the C-terminal loop of SNase plays
an important role in protein stability and conforma-
tional integrity. During protein unfolding ⁄ refolding,
the addition of 2.5 protons (both E75 and E129 are
the targets of protonation) results in SNase unfolding,
with refolding at an early stage through the formation
of a SNase(1–139) fragment in which the tryptophan
at position 140 is needed for formation of the native
structure.
Experimental procedures
Materials
Luria–Bertani broth and isopropyl thio-b-d-galactoside
were purchased from USB (Cleveland, OH). Salmon testes
DNA and some analytical grade chemicals such as EDTA,
Tris ⁄ HCl, CaCl
2
, NaCl and mineral oil were obtained from
Sigma (St Louis, MO, USA). Salmon testes DNA for the
enzyme activity test was used without further purification.
Guanidine hydrochloride and dNTPs were purchased from
Boehringer (Mannheim, Germany). Ethanol (> 99%) was
obtained from Panreac (Barcelona, Spain). Urea was a
product of Acros (Pittsburgh, PA, USA). The Quick-
change
TM

kit containing Pfu DNA polymerase, 10 · reac-
tion buffer and DpnI restriction enzyme was purchased
from Stratagene (La Jolla, CA, USA). Water used for these
experiments was deionized and distilled.
PCR site-directed mutagenesis
The wild-type SNase nuc gene (originally obtained from
D. Shortle, the Johns Hopkins University, Baltimore, MD,
USA) was cloned into pTrc-99A, which was used to trans-
form Escherichia coli strain JM105. Plasmid DNA was
purified by the alkaline lysis method (Gibco-BRL, Gaithers-
burg, MD, USA; GFX
TM
kit), and stored at )20 °C before
being subjected to mutagenesis. Two complementary 33-mer
primers that included the alanine codon at positions 140 or
133 were designed and synthesized (Life Technologies,
Rockville, MD, USA). Single-point mutations were made
by site-directed mutagenesis to generate W140A and
K133A. Truncated proteins W140O (the segment between
positions 140 and 149 of the wild-type protein was removed)
and E142O (the segment between positions 142 and 149 of
the wild-type protein was removed) were generated by using
suitable complementary primers. For site-directed mutagen-
esis, a 10 · reaction buffer (Stratagene; QuickChange
TM
kit)
was mixed with 1.5 lL dsDNA template, 1.2 lL of a pair of
complementary oligonucleotides, 1 lL10mm each dNTP
and double-distilled water to a final volume of 50 lL. Then
1 lL Pfu DNA polymerase was added to the solution, and

the mixture was overlaid with 30 lL mineral oil. A PCR
consisting of 16 cycles of 50 °C (1.5 min), 68 °C (14 min),
and 94 °C (1 min) was performed using a PerkinElmer 480
thermal cycler (Wellesley, MA, USA). The wild-type DNA
template was then digested by adding 1 lL DpnI restriction
enzyme to the PCR mixture and incubating at 37 °C for
1 h. Then 10 lL of the reaction mixture (containing undi-
gested mutant plasmid) was used to transform 100 lL
B
A
Fig. 4. Global segment interactions and the folding profiles in wild-
type SNase. (A) W140, in loop 1 interacts with loop 2 and loop 3
which forms a ‘lower neck’ network area in maintaining protein ter-
tiary structure. State 1 denotes the nascent polypeptide fragment.
(B) The fragment folding pathway induced via the SNase(1–139)
fragment and formed to its minimum energy native state via the
addition of either tryptophan at position 140 or fragment 140–149.
Staphylococcal nuclease refolding Z. Su et al.
3964 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS
competent JM105 cells. The mixture was incubated on ice
for 1 h, and at 42 °C for 2 min, followed by 2 min incuba-
tion on ice. After transformation, 800 lL Luria–Bertani
medium was added, and the mixture incubated at 37 °C for
1 h. Transformed cells were selected on ampicillin plates,
and mutant DNA was isolated from the resulting colonies.
Mutant plasmids were then identified by BamHI and NcoI
restriction digestion, and their sequences confirmed by
DNA sequencing.
DNA sequencing
Plasmid DNA was isolated with the GFX

TM
Micro Plas-
mid Prep Kit (Amersham Pharmacia Biotech, Piscataway,
NJ, USA), and the resulting dsDNA was mixed with 8 lL
BigDye
TM
master mix (BigDye
TM
Terminator Ready Reac-
tion Kit; Applied Biosystems, Foster City, CA, USA) and
3.2 pmol sequencing primer. The final solution was mixed
with deionized water to a final volume of 20 lLina
0.5-mL thin-walled PCR tube and overlaid with 40 lL light
mineral oil. DNA sequencing was performed by cycle
sequencing using 25 cycles of 96 °C for 30 s, 50 °C for
15 s, and 60 °C for 4 min in a PerkinElmer 480 thermal
cycler. The extension products were purified by a Centri-
Sep
TM
spin column chromatography (Princeton Separa-
tions, Adelphia, NJ, USA) to remove unincorporated dye
terminators. Template suppression reagent (5 lL; PE
Applied Biosystems, Foster City, CA, USA) was mixed
with purified extension products. The samples were heated
at 95 °C for 2 min and then chilled on ice. Capillary elec-
trophoresis was performed using an ABI PRISM 310 Gen-
etic Analyzer (PE Applied Biosystems) fitted with a 47-cm
capillary containing POP-6 polymer. The mutant sequences
(positions 1–149 for mutant proteins or 1–139 and 1–141
for truncated proteins) were compared with that of the

wild-type and confirmed to have the correct mutant
sequences.
Protein purification
Escherichia coli JM105 carrying recombinant plasmids were
grown in Luria–Bertani broth containing 100 lg ÆmL
)1
ampicillin at 37 °C. Protein expression was induced by
adding isopropyl thio-b-d-galactoside. The cells were har-
vested after 4 h of incubation and suspended in chilled
buffer A (6 m urea, 0.05 m Tris, 0.2 m NaCl, pH 9.2, fil-
tered through a 0.45 lm membrane). Proteins were collec-
ted after two alcohol precipitations and stored in buffer B
(6 m urea, 0.05 m Tris, pH 9.2, filtered through a 0.45 lm
membrane). The recombinant proteins were purified by
cation-exchange chromatography (washed CM-25 ion-
exchange gel column). The proteins were dialysed after
purification for 2 days at 4 °C against phosphate buffer
(25 mm NaH
2
PO
4
⁄ 50 mm NaHPO
4
⁄ 200 mm NaCl, pH
adjusted to 7.0) and were then lyophilized. The average
yield of recombinant proteins was  25 mgÆL
)1
. SNase
purity was investigated by SDS ⁄ PAGE. The gel was
stained with Coomassie blue and analyzed by densitome-

try, revealing protein purity of greater than 85%. Protein
concentration was determined by measuring the absorption
coefficient of each mutant by the method of Gill & von
Hippel [27].
CD measurements
CD was performed on wild-type protein and mutants
using a Jasco model J-720 spectropolarimeter. The spectra
were measured between 200 and 320 nm. Wild-type and
mutant proteins were dissolved in phosphate buffer
(25 mm NaH
2
PO
4
⁄ 50 mm NaHPO
4
⁄ 200 mm NaCl, pH
adjusted to 7.0) at a concentration of 0.5 mgÆmL
)1
. Spec-
tra were obtained as the average of five successive
scans with a bandwidth of 1.0 nm and a scan speed of
20 nmÆmin
)1
.
Steady-state tryptophan fluorescence
measurements
Measurements were made with a LS-50B spectrometer
(PerkinElmer). Samples were dissolved in phosphate buffer
at a concentration of 0.4 mgÆmL
)1

. Excitation was set at
298 nm, and emissions were observed at 350 nm. The fluor-
escence spectra were measured between 300 and 550 nm
with a scanning speed of 150 nmÆs
)1
and an excitation slit
of 5.0 nm.
Calorimetric measurements
Thermal analysis of protein denaturation was performed
with DSC (a model 6100 Nano II; Calorimetry Sciences
Corp., Provo, UT, USA). Lyophilized wild-type and
mutant SNase were dissolved in phosphate buffer at a
concentration of 2 mg ÆmL
)1
. Samples were first sonicated
for 15 min. Then 1 mL buffer or sample was loaded into
a clean reference or sample cell, respectively, ensuring that
the samples were free of air bubbles. Samples were heated
from 20 °Cto75°C under 3 atm at a heating rate of
1 °CÆmin
)1
. The melting point (T
m
) of protein tested was
directly obtained from the DSC curve. The enthalpy
change (DH
cal
) of each protein was calculated by integra-
tion of the curve covering area (T
m

was taken as the
curve peak point) using origin software.
Acknowledgements
This work is partially supported by a grant (NSC-
92-2311-B-001) from the National Science Council,
Taiwan, R.O.C. and the theme project of Academia
Sinica, Taipei, Taiwan, R.O.C.
Z. Su et al. Staphylococcal nuclease refolding
FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS 3965
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