In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 BSL2.. Recently, the crystal structure of house fly digestive lysozyme was solve
Trang 1lysozyme from cow stomach
Yasuhiro Nonaka1, Daisuke Akieda1, Tomoyasu Aizawa1, Nobuhisa Watanabe1,2, Masakatsu
Kamiya3, Yasuhiro Kumaki1, Mineyuki Mizuguchi4, Takashi Kikukawa1, Makoto Demura3and
Keiichi Kawano1
1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan
2 Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan
3 Division of Molecular Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan
4 Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
C-type lysozyme (EC 3.2.1.17), represented by hen
egg-white lysozyme (HEWL), is one of the most
well-known enzymes It has been found in various
ver-tebrates, arthropods, and some other metazoa It
cata-lyzes the hydrolysis of the b-1,4-glycoside linkage
between N-acetylglucosamine and N-acetylmuramic
acid of peptidoglycan, and thus breaks the bacterial
cell wall [1] Most c-type lysozymes reported thus far
are considered to play a role in defense against
bacte-rial infection It was proposed that the bacteriolytic
activity of lysozymes is also used for digestion in some
species
In artiodactyl ruminants, which feed on plants, the
foregut chamber has evolved to digest cellulose
efficiently [2–4] They recruit bacteria that ferment
cellulose in the foregut The bacteria are broken down
by lysozyme in the true stomach, and the digested com-ponent is then absorbed in the intestine The acquisi-tion of digestive lysozyme is well known as a case of convergent evolution [4] In addition to artiodactyla, many other animals, such as a folivorous monkey (col-obus) and a bird (hoatzin), as well as the house fly, are known to have digestive c-type lysozymes [5–7] Those folivorous animals obtain nourishment from plant material in a similar manner to artiodactyla House fly larvae feed on bacteria growing in decomposing mate-rial, and digest the bacteria with lysozyme
According to phylogenetic analyses, each phyloge-netic group has independently adapted its defensive lysozyme for digestion [7,8] Interestingly, common
Keywords
lysozyme; molecular evolution; protease
resistance; structural stability; X-ray
crystallography
Correspondence
K Kawano, Graduate School of Science,
Hokkaido University, North 10, West 8,
Kita-ku, Sapporo, Hokkaido 060 0810,
Japan
Fax: +81 11 706 2770
Tel: +81 11 706 2770
E-mail: kawano@mail.sci.hokudai.ac.jp
(Received 12 November 2008, revised 22
January 2009, accepted 4 February 2009)
doi:10.1111/j.1742-4658.2009.06948.x
In ruminants, some leaf-eating animals, and some insects, defensive lyso-zymes have been adapted to become digestive enlyso-zymes, in order to digest bacteria in the stomach Digestive lysozyme has been reported to be resis-tant to protease and to have optimal activity at acidic pH The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2) Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH The high stability in acidic solution could be related to pepsin resistance, which has been previ-ously reported for BSL2 The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could pro-vide structural stability, and thus resistance to pepsin It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin
Abbreviations
BSL2, bovine stomach lysozyme 2; DSC, differential scanning calorimetry; HEWL, hen egg-white lysozyme.
Trang 2properties, e.g low optimal pH and resistance to
pro-tease, are shared by digestive lysozymes from different
organisms [6,8–10] Furthermore, ruminant and
colo-bus lysozymes share similarities in amino acid
sequence, and this is unlikely to have occurred by
random drift, suggesting convergent (or parallel)
amino acid replacements [7] These functional and
structural similarities could have resulted from
adap-tation to severe gastric conditions However, the
molecular bases for such adaptations remain to be
investigated Recently, the crystal structure of house
fly digestive lysozyme was solved, explaining the
mech-anism underlying the acidic pH optimum [11] The pKa
values of the catalytic residues are lowered by
neigh-boring residues, resulting in the acidic pH optimum
No experimental three-dimensional structure of
ver-tebrate digestive lysozyme has been reported thus far
It would be useful to understand the structural bases
for the adaptation by comparing this lysozyme with
house fly digestive and other nondigestive lysozymes
In this study, we obtained recombinant bovine
stom-ach lysozyme 2 (BSL2), the most highly expressed
lyso-zyme in the cow stomach X-ray crystallography and
some other experiments were performed to determine
how this lysozyme has acquired the properties
mentioned above We also discuss the significance of
the probable convergent amino acid replacements
Results
X-ray crystallography of BSL2
The crystal structure of BSL2 is shown in Fig 1A, and
the data collection, processing and refinement statistics
are summarized in Table 1 BSL2 was crystallized in
the space group P212121 The structure was refined at
1.5 A˚ to an R-factor of 17.8% and an R-free of
22.1% The average B-value for all protein atoms is 10.17 A˚2, and that for all main chain atoms is 9.25 A˚2 The electron density map was sufficiently clear to build
a molecular model, and most of the side chain confor-mations were determined unequivocally, although some residues showed multiple conformers
This lysozyme is composed of an a-domain and a b-domain, both of which are common in the previ-ously reported structures for other c-type lysozymes The a-domain is composed of four a-helices (A–D), and the b-domain is composed of a large loop and a three-strand antiparallel b-sheet Figure 1B is a super-imposition of the main chain conformations of BSL2, human lysozyme, HEWL, and house fly midgut
Fig 1 (A) Ribbon model of BSL2 (Protein
Data Bank ID: 2Z2F) in which a-helices are
sequentially labeled from A to D The
struc-ture is shown in rainbow colors from the
N-terminus to the C-terminus The figure
was produced using MOLFEAT (FiatLux,
Tokyo, Japan) (B) Superimposition of the C a
conformation of BSL2 (red), human
lyso-zyme (green, 1JSF), HEWL (blue, 1DPX),
and house fly midgut lysozyme (yellow,
A chain of 2FBD) The broken-line circle
represents the loop region following the
C-helix The figure was produced using
MOLMOL [50].
Table 1 Data collection, processing and refinement statistics Data collection
Cell constants (A ˚ )
Refinement data
Rmsd from ideal values
a Values in parentheses are for the last resolution shell.
Trang 3lysozyme The rmsd between BSL2 and human
lyso-zyme, calculated using the backbone atoms in the
a-helices, is 0.38 A˚, that between BSL2 and HEWL is
0.35 A˚, and that between BSL2 and house fly lysozyme
is 0.79 A˚ The backbone structure of BSL2 is closer to
that of the vertebrate nondigestive lysozyme than to
that of insect digestive lysozyme
pH dependence of the lytic activity of BSL2
The digestive lysozymes reported thus far tend to have
a pH optimum at acidic pH, whereas nondigestive
lysozymes have a broad optimum at neutral pH [8,9]
The relative lytic activities of recombinant BSL2 and
commercial HEWL at pH 4–7 are shown in Fig 2
The pH optimum of BSL2 was about 5, whereas that
of HEWL occurred at pH values higher than 6 BSL2
exhibited less activity than HEWL, even at the optimal
pH of BSL2 At pH 7, BSL2 showed almost no lytic
activity
Structural stability of BSL2 in acidic conditions
Digestive lysozymes need protease resistance to
main-tain their lytic activity in the stomach As shown in
Fig 3, BSL2 is more resistant than HEWL to pepsin
Pepsin readily digested HEWL in acidic conditions
with physiological ionic strength (150 mm NaCl),
whereas BSL2 remained intact after 4 h This result
corresponded to that for natural BSL2 from bovine
stomach, based on residual activity [9]
In one report, protease resistance was correlated with protein thermostability [12] To evaluate the structural stability of BSL2, denaturant-induced unfolding and thermal unfolding were monitored Figure 4 shows the guanidinium hydrochloride-unfolding curves of BSL2 and HEWL, as determined by CD ellipticity at 222 nm, indicating the disruption of the native structure The parameters derived from these unfolding curves are shown in Table 2 At pH 6.0, BSL2 and HEWL were similar in their midpoints (Cm), Gibbs free energies without denaturant (DGw), and m values indicative of cooperativity At pH 2.0, in contrast, BSL2 unfolded at
a higher concentration of guanidinium hydrochloride than HEWL The Gibbs free energy of BSL2 at low
pH was much greater than that of HEWL, indicating the high conformational stability of BSL2 The transi-tion temperatures (Tm) and unfolding enthalpy values (DHu) at pH 2.0, obtained by thermal unfolding experi-ments using differential scanning calorimetry (DSC), are also summarized in Table 2 BSL2 unfolded at a higher temperature and had a greater DHu value, also indicating greater structural stability
Hydrogen exchange properties were monitored by 1D 1H-NMR at pH 1.9, to compare the conforma-tional flexibilities of BSL2 and HEWL (Fig 5) Gener-ally, there are few or no peaks around 10 p.p.m., except for the peaks of tryptophan indole hydrogen atoms Both BSL2 and HEWL have six tryptophan residues, and five peaks appear around 10 p.p.m for both proteins In the spectra of HEWL, most of the indole hydrogen peaks diminished rapidly within 30–60 min, and only the peak at 10.3 p.p.m remained after a 120 min exchange In the spectra of BSL2, three peaks were observed after the 30 min exchange, and decreased gradually In particular, the peak of Trp64 in BSL2 diminishes more slowly than that of the corresponding residue, Trp63, in HEWL The tryp-tophan residues whose peaks diminished rather slowly could exist in rigid and unexposed regions
Fig 2 Bacteriolytic activities of BSL2 (gray bars) and HEWL (white
bars) at different pH values, ionic strength 0.1, and 25 C The
rela-tive activities are expressed by taking the activity of HEWL at
pH 7.0 as 1.0.
A B
C
Fig 3 SDS ⁄ PAGE of pepsin-treated BSL2 and HEWL with (A)
0 m M NaCl (B) 150 m M NaCl, and (C) 500 m M NaCl Aliquots of the solution were sampled at intervals of 1 h M is the marker lane.
Trang 4Although BSL2 has an acidic optimal pH, the relative
activity level is lower than or comparable to that of
HEWL, even at acidic pH (Fig 2) BSL2, like many acidophilic proteins [13–15], possesses a greater num-ber of acidic residues than nondigestive lysozymes (Table 3) An increase in acidic residues would result
in low lytic activity, because the electrostatic attraction between the lysozyme and the negatively charged bac-terial membrane becomes weaker, especially at neutral
pH BSL isozymes are considered to function below
pH 6 in nature [9] It is likely that BSL2 has lost lytic activity at neutral pH and retains it below pH 6
In the case of house fly digestive lysozyme, the crys-tallographic analysis and catalytic activity experiments indicated that the catalytic residues have lower pKa
values than those of HEWL, and thus the optimal pH
is shifted to the acidic range [11] Using the crystallo-graphic structures, we calculated the pKa values of the
Fig 4 Guanidinium hydrochloride-induced unfolding curves of
BSL2 (circles) and HEWL (triangles) monitored by CD at (A) pH 2.0
and (B) pH 6.0 The apparent fractions of unfolding protein, fapp,
were plotted against the concentration of guanidinium
hydrochlo-ride The lines are the transition curves estimated by the nonlinear
least squares method.
Table 2 Thermodynamic parameters for guanidinium
hydrochlo-ride-induced and thermal unfolding.
Guanidinium hydrochloride-induced unfolding
Thermal unfolding
DH (kJÆmol)1) a 406.4 386.4
a The unfolding enthalpies at transition temperature Tm.
A
B
p.p.m.
p.p.m.
W63 W34 W108
Fig 5 1D 1 H-NMR spectra of (A) BSL2 and (B) HEWL in 95%
H2O ⁄ 5% D 2 O (thick lines) and after 30, 60 or 120 min of hydro-gen–deuterium exchange in 100% D 2 O (thin lines) The spectra were acquired at pH 1.9.
Trang 5catalytic residues Glu35 and Asp52 (numbering for
HEWL), for BSL2 and other lysozymes, with
prop-ka2.0 [16] The predicted pKa values were 6.15 and
4.27 for BSL2, 5.93 and 4.20 for HEWL, and 4.89 and
3.84 for house fly lysozyme Although these values do
not agree completely with the experimental results [11],
the acidic shifts of the pKa values for house fly
lyso-zyme are well predicted The calculated pKa values for
BSL2 are not reduced as compared to those for
HEWL Glu35 in BSL2 is surrounded by hydrophobic
residues, as it is in HEWL, and this results in the high
pKa, whereas the polarity of Thr110 reduces the pKa
for house fly lysozyme In the case of Asp52, the pKa
is modulated by the hydrogen bond network There
are hydrogen bonds formed by Asp52, Asn46 and
Asp48 in HEWL House fly lysozyme has an
aspara-gine at position 48, and the absence of the negative
charge should reduce the pKa of Asp52 as compared
to HEWL [11] Asn46 in BSL2 is distant from Asp52,
and the absence of this hydrogen bond network would
reduce the pKa However, Asp52 in BSL2 is more
exposed to solvent than that in HEWL, and this raises
the pKa As a result, the calculated pKa values for
BSL2 were comparable to those for HEWL The result
suggests that the catalytic activity of BSL2 is not
adapted to acidic conditions, unlike the case with
house fly lysozyme
BSL2 and other vertebrate digestive lysozymes have
been reported to be resistant to pepsin digestion, as is
also shown in Fig 3 The efficiency of peptide bond
fission by protease reflects the conformational
flexibil-ity of the polypeptide substrate [12,17,18] The
correla-tion between structural rigidity and stability has been
reported for many proteins [19–22] The high
confor-mational stability of BSL2 as compared to HEWL
(Table 2) suggests greater structural rigidity The
higher rigidity was also suggested by the hydrogen
exchange experiment (Fig 5) Trp64 in BSL2 is
pro-tected, whereas Trp63 in HEWL is not This residue
exists in the b-domain, and is oriented to the interface
between the two domains Therefore, the interface of
BSL2 is less susceptible to unfolding than that of
HEWL These results support the notion that confor-mational rigidity protects BSL2 from pepsin digestion Because the house fly lysozyme is resistant to cathep-sin D, a protease from the house fly midgut [5], the house fly midgut lysozyme would have structural stability and rigidity similar to that of BSL2 As observed for thermophilic enzymes, an increase in con-formational rigidity often leads to a reduction in enzy-matic activity [22–24] The lower lytic activity of BSL2 (Fig 2) may also be caused by the increased rigidity, and not only by the increased negative charge
The numbers of positive and negative charges differ among these lysozymes (Table 3) The surfaces of HEWL and human lysozyme are predominantly posi-tively charged A lysozyme covered with posiposi-tively charged surfaces will have a loose structure, because electrostatic repulsion significantly increases on the molecular surface BSL2 has a negatively charged b-domain and a positively charged a-domain The electrostatic repulsion on the surface will be weaker, and this could contribute to the higher stability There are fewer charged residues on the surface of the house
fly lysozyme, and the electrostatic repulsion will be smaller The house fly lysozyme may have achieved structural stability by decreasing the positively charged residues
The increase in acidic residues is also expected to result in an increase in the number of salt bridges The numbers of the salt bridges in BSL2 and HEWL, how-ever, are comparable (Table 3) It is noteworthy that BSL2 contains a complex salt bridge (Glu83–Lys91– Glu86) that is absent in the three other lysozymes A triangular salt bridge formed by two acidic residues and one basic residue can be more strong than the sum of simple salt bridges [25–27] The loop located between Glu83 and Lys91 connects the b-domain and the a-domain In the case of calcium-binding lysozyme, calcium binding at this loop stabilizes the native struc-ture [28,29] By analogy, the electrostatic interaction at this loop is considered to contribute to the overall structural stability
The overall structures of these lysozymes are very similar (Fig 1B), and the numbers of hydrogen bonds are comparable (Table 3) A marked difference is observed in the region from the C-terminus of the C-helix to the following loop, residues 100–103 in HEWL (Fig 1B) The C-helices of human lysozyme and HEWL are terminated at residue 101 followed by proline or glycine, which can destabilize the a-helix [30] BSL2 and house fly lysozyme lack this proline or glycine residue, and thus the C-helices are longer and the following loops are shorter than those of HEWL and human lysozyme This would prevent pepsin
Table 3 Comparison of structural parameters among lysozymes.
No of charged residues
Hydrogen bonds ⁄ residue 0.97 0.95 0.96 0.89
Trang 6digestion, because there are proteolytic sites for pepsin
in this loop for HEWL and human lysozyme [18,31]
The amino acid replacements at positions 14, 21, 50,
75 and 87 were considered to be significant for the
adaptation of digestive lysozyme, on the basis of the
analyses using vertebrate digestive and nondigestive
lysozyme sequences [7,32] No remarkable difference,
such as the alteration of hydrogen bonds, is found at
these positions between BSL2 and human lysozyme,
except at residue 21 The side chain of Lys21 in BSL2
forms hydrogen bonds with the side chains of Tyr20
and Ser101, whereas the side chain of Arg21 in human
lysozyme hydrogen-bonds to the backbone carbonyl
oxygens of Val100 and Asp102 As discussed above,
the region that includes residues 100–102 could be
associated with resistance to pepsin The replacement
of residue 21 could also be an adaptation to stabilize
this region
Experimental procedures
Expression and purification of BSL2
In an Escherichia coli expression system, removal of an
extra methionine residue at the N-terminus does not take
place in the case of lysozyme [33] We obtained
recom-binant BSL2 with a perfect sequence using the
methylo-trophic yeast Pichia pastoris, basically as described by
Digan et al [34]
The cDNA was ligated to the expression vector pPIC3
(Invitrogen, Carlsbad, CA, USA) To secrete BSL2 into the
culture, we incorporated the native signal sequence of
BSL2 The plasmid was linearized by SalI, and transformed
into P pastoris GS115 by electroporation Genotypic
selec-tion and phenotypic screening were performed on a
mini-mal dextrose plate (1.34% yeast nitrogen base, 4· 10)5%
biotin, 1% dextrose, and 1.5% agar) and on a minimal
methanol lysoplate (1.34% yeast nitrogen base, 4· 10)5%
biotin, 0.061% Micrococcus lysodeikticus, and 1.5% agar,
in 10 mm potassium phosphate buffer, pH 5.0), as
previ-ously reported, except for pH and buffer concentration
[35] Colonies on a minimal dextrose plate were inoculated
onto a minimal methanol lysoplate, and 200 lL of
metha-nol was spread on the plate cover and incubated at 30C
for about 1–3 days The radius of the translucent plaque
around the colony was measured as an indicator of the
colony’s lysozyme expression level
P pastorisfor BSL2 expression was cultivated using a jar
fermenter with high-density fermentation [36–38] To avoid
proteolysis, we recovered the culture after induction for
48 h To purify recombinant lysozyme using cation
exchange chromatography, the supernatant of the culture
was diluted so that the electrical conductivity was decreased
to < 5 mSÆcm)1 The diluted supernatant was filtered
through a nitrocellulose membrane The supernatant was loaded onto an SP-Sepharose Fast Flow column (300 mL) (GE Healthcare, Piscataway, NJ, USA) equilibrated with
50 mm sodium acetate buffer (pH 4.8), and the adsorbed proteins were eluted with 50 mm sodium acetate buffer with
1 m NaCl (pH 4.8) The elution was monitored by absor-bance at 280 nm The sample solution was dialyzed with
50 mm sodium acetate buffer (pH 4.8) to decrease electrical conductivity After dialysis, the sample was loaded onto an SP-Sepharose Fast Flow column equilibrated with 50 mm sodium acetate buffer (pH 4.8), and eluted with a salt linear gradient of 50 mm sodium acetate buffer with 1 m NaCl (pH 4.8) The main peak fraction was dialyzed with 20 mm
NH4HCO3and freeze-dried
Assay of lytic activity The lytic activities of BSL2 and HEWL against M lys-odeikticus were estimated using the turbidimetric method [39] Lyophilized M lysodeikticus was purchased from Sigma-Aldrich (St Louis, MO, USA) Suspensions of
M lysodeikticus were prepared in sodium acetate (for pH 4 and 5) and sodium phosphate (for pH 6 and 7) buffer The ionic strength of each buffer was adjusted to 0.1 [40] Lyso-zyme solution and M lysodeikticus suspension were mixed, and the decrease in absorbance was monitored at 540 nm with a thermostatically controlled cell holder at 25C The relative activity was calculated from the speed of the absor-bance decrement
Pepsin digestion Pepsin was obtained from Sigma-Aldrich HEWL was obtained from Seikagaku Corp (Tokyo, Japan) Lysozymes were dissolved in 10 mm HCl (pH 2), and the final protein concentration was 0.5 mgÆmL)1 The digestion experiment was carried out in the presence of pepsin at 37C The aliquots were sampled at intervals of 1 h and then frozen until electrophoresis
X-ray crystallography
A crystal of BSL2 was obtained by the vapor diffusion (sit-ting drop) method, using 0.1 m sodium Hepes buffer at
pH 7.5, containing 0.2 m NaCl and 30% 2-methyl-2,4-penta-nediol The space group of the crystal was P212121, with cell dimensions a = 31.257 A˚, b = 56.065 A˚, and c = 64.050 A˚ There is one monomeric molecule in an asymmet-ric unit The X-ray diffraction data of BSL2 were collected from a single crystal at 93 K, using a MicroMAX-007 generator (Rigaku, Tokyo, Japan) and an R-AXIS IV++ detector (Rigaku) The reflections were processed with the program hkl-2000 [41] The I⁄ r(I) in the last resolution shell (1.55–1.50) was 17.272 The resolution was limited by the
Trang 7acceptance of the detector The limit at the edge of the
detec-tor using an 80 mm crystal-to-film distance is approximately
1.5 A˚ resolution The structure was solved by the molecular
replacement method, using the program molrep [42]
pack-aged in ccp4 [43] The structure of recombinant human
lyso-zyme (Protein Data Bank code: 1LZ1) [44] was used as the
search model The structure was refined using the program
refmac5 [45] in the ccp4 suite, and was visually inspected
using coot [46] Water molecules were found by the
func-tions in refmac5 and coot, and were checked visually using
coot A sodium ion was added to the model as judged by the
electron density, coordination number, and interatomic
dis-tance The structure was deposited in the Protein Data Bank
under the code 2Z2F
Analysis of structural features
A salt bridge in Table 3 was defined as a negative residue
and a positive residue with an interatomic distance of
< 4.0 A˚ The hydrogen bonds were detected using the
what if web interface with the following criteria: maximal
distances of 3.5 A˚ for donor–acceptor and 2.5 A˚ for
hydrogen–acceptor, and minimal angles of 60 for donor–
hydrogen–acceptor and 90 for hydrogen–acceptor–X
Water-mediated hydrogen bonds were not included
CD
CD at 222 nm was measured with a Jasco J-725
spectro-polarimeter (Japan Spectroscopic, Tokyo, Japan), using
optical cells with path length of 1 mm The guanidinium
hydrochloride-induced unfolding experiment was carried
out at 298 K using 50 mm KCl⁄ HCl buffer at pH 2.0, and
50 mm sodium phosphate buffer at pH 6.0 The
concentra-tion of guanidinium hydrochloride was determined by the
difference between the refractive indices of guanidinium
hydrochloride solution and guanidinium hydrochloride-free
solution The protein concentration was 8–10 lm The
unfolding curves were fitted to the following equation:
DG = – RTlnK = DGw– mC, where DG and DGware the
Gibbs free energy with denaturant and that without
denaturant respectively, and R, T, K, m and C are the gas
constant, absolute temperature, equilibrium constant,
cooperativity index, and denaturant concentration,
respectively
DSC
DSC was carried out using VP-DSC (MicroCal,
Northamp-ton, MA, USA), at a scan rate of 1.0 KÆmin)1 Sample
solution was prepared with reference buffer 50 mm
glycine-HCl at pH 2.0 To extend the temperature range, all DSC
measurements were performed under a pressure of 2.0 atm
The protein concentration and pH were confirmed after the
scan The DSC curves were analyzed to obtain the transi-tion temperatures (Tm) and unfolding enthalpies (DHu) [47]
Hydrogen–deuterium exchange experiment Hydrogen–deuterium exchange was measured by 1D 1 H-NMR performed on a Bruker 500 MHz instrument (Bruker BioSpin, Rheinstetten, Germany), with a cryogenic probe and a JEOL ECA-600 instrument (JEOL, Tokyo, Japan) The exchange was initiated by dissolving protein that had been lyophilized with pH-adjusted buffer (pH 1.9) in D2O
to give a final protein concentration of 0.3 mm in 50 mm sodium phosphate The sample was incubated at 298 K A total of 32 scans of each sample were collected at 30 or
60 min intervals To acquire the spectra before hydrogen exchange, lysozyme solution was subjected to 1H-NMR in the same buffer with 95% H2O⁄ 5% D2O The peaks of unexchangeable hydrogens were used to normalize inten-sity The peaks of indole hydrogens were assigned on the basis of the BMRB database (bmr1093 and bmr4562 for HEWL and bmr76 for human lysozyme were used), and using proshift [48], a chemical-shift prediction tool
Estimation of protein concentration The protein concentrations were estimated spectrophoto-metrically by following the extinction coefficients at 280 nm for a 1% solution in a 1 cm cell: E = 28.4 for BSL2, and
E= 26.5 for HEWL, estimated using protparam [49]
Acknowledgements
This study was supported by the Program for the Pro-motion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan We thank the staff
of the High-Resolution NMR Laboratory, Graduate School of Science, Hokkaido University, for the NMR measurements, Professor I Tanaka, Graduate School
of Life Science, Hokkaido University, for the X-ray crystallography, and Emeritus Professor K Nitta, Graduate School of Science, Hokkaido University, for helpful advice
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