The electrostatic interactions between charged residues therefore make significant contributions to protein stability in unfolding.. We hypothesized that only certain charged amino acids
Trang 1amino acid residues in staphylococcal nuclease unfolding Hueih-Min Chen1, Siu-Chiu Chan1, King-Wong Leung1, Jiun-Ming Wu1, Huey-Jen Fang1
and Tian-Yow Tsong2,3
1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan
2 Institute of Physics, Academia Sinica, Taipei, Taiwan
3 Department of Biochemistry, University of Minnesota College of Biological Sciences, St Paul, MN, USA
Staphylococcal nuclease (SNase) is a globular protein
that consists of 149 amino acids with a molecular mass
of about 16 kDa This protein lacks disulfide bonds in
its native structure Because of the absence of covalent
bonds, electrostatic interactions, hydrophobic forces,
van der Waals’ forces and hydrogen bonds are the only
significant forces acting during protein denaturation
and renaturation Thus SNase provides a simplified
model for studying protein folding In addition, this
protein contains a large number of charged amino
acids, including 20 negatively charged residues and 32
positively charged residues (more than 35% of the 149
amino acids) When SNase is unfolded by pH titration
in acid (pH 7 to pH 2), the transition point during
unfolding occurs around pH 4 [1], which corresponds
to the pKa of glutamic acid or aspartic acid [2] The
electrostatic interactions between charged residues
therefore make significant contributions to protein
stability in unfolding Because electrostatic force is inversely proportional to the distance between charges,
we chose to examine the interaction of oppositely charged residues that were predicted to be in close proximity to each other We hypothesized that only certain charged amino acids at particular locations in SNase are critical for stabilizing local protein structure Recent developments in the cloning and expression
of SNase and its mutants in Escherichia coli have allowed this protein to be used as a model for biophys-ical studies of protein folding kinetics [1–3], rotational mobility of tryptophan residues [4], the effects of mutations on its structure and stability [5–7] and calorimetric analysis [6–8] Our previous studies indica-ted the presence of significant electrostatic interactions between charged amino acids in the protein’s native state Specifically, we identified a local stable segment surrounding the glutamic acid at position 75 formed
Keywords
staphylococcal nuclease; local stability; key
acidic amino acid; unfolding
Correspondence
H.-M Chen, Institute of BioAgricultural
Sciences Academia Sinica, Taipei, Taiwan
Fax: +886 2 2788 8401
Tel: +886 2 2785 5696 extn 8030
E-mail: robell@gate.sinica.edu.tw
(Received 3 May 2005, revised 3 June
2005, accepted 13 June 2005)
doi:10.1111/j.1742-4658.2005.04816.x
Staphylococcalnuclease is a single domain protein with 149 amino acids It has no disulfide bonds, which makes it a simple model for the study of pro-tein folding In this study, 20 mutants of this propro-tein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid Using differential scanning microcalorimetry in ther-mal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower DHcal values than the wild-type protein Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly resi-due has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability Two local stable areas formed by the charge–charge interactions around E75 and E129 with particular positively charged amino acids are thus iden-tified as being significant in maintenance of the three-dimensional structure
of the protein
Abbreviations
SNase, staphylococcal nuclease; WT, wild-type.
Trang 2by the interactions of E75 with H121 and K97 It is
believed that a small number of key amino acid
seg-ments play major roles in generating these forces that
most likely support the protein’s structural frame If
one observes a native protein as it refolds from a
ran-dom unfolded state, the efficiency of refolding
indi-cates that some energetically interactive areas within
the protein must form preferentially due to relatively
strong forces of attraction Based on this model,
pro-tein refolding can be considered a process of
thermo-dynamic nucleation that includes an essential step in
which specific stable segments are formed We have
been trying to find these stable segments in SNase: one
of these may be located in the domain among E75,
K9, Y93 and H121 An area formed by these
inter-actions can be imagined as a ‘hand’ clamped tightly
around the ‘neck’ of the whole protein This clamped
area was first suggested by Privalov’s group [8] with
two strong interactions in SNase: an electrostatic
inter-action between E75 and H121 and a hydrogen bond
between D77 and T120 These bonds play significant
roles in the cooperation of the two subdomains: a
C-terminal a-helix and a b-barrel [9,10] In our E75G
mutant, X-ray diffraction results had shown that this
locally stable network is absent due to the loss of the
hydrogen bonding among E75 and K9, Y93 and H121
[11] Instead, one water molecule hydrogen bonds to
Y93 and H121 Both G75 and K9 are completely
out-side of this local bonding area Thus, the two
sub-domains are unable to interact cooperatively This
structural rearrangement may allow E75G mutant to
unfold with only half the enthalpic energy of the
wild-type protein If E75G mutant was to be denatured by
a change in pH, we further predicted that its
C-ter-minal a-helix would unfold through the I state
(inter-mediate state) [8,12] and the flexible b-barrel would be
subsequently destroyed
In addition to E75 forming a local stable area in
SNase, in this study we searched for the existence of
other local stable segments that may play similar key
roles in sustaining protein native structure We used
site-directed mutagenesis to create 20 distinct glycine
substitution mutants of single negative charged
resi-dues in order to dissect elements important for SNase
stability We found that E75 and E129 play the role
for retaining SNase tertiary structure In order to
dis-cern whether it is due to the absence of an acidic
resi-due (glutamic acid or aspartic acid), or the presence of
glycine which destabilizes SNase secondary structure,
both E75Q and E129Q were created The results
revealed that only a little influence on the stability of
protein tertiary structure is due to the particular
sub-stitution of glycine at positions 75 and 129 The
charge–charge interactions around these two local areas (around E75 and E129) are the most significant factors for maintaining the whole protein stability
Results
CD spectra Secondary structures of SNase and its mutants were determined by CD with spectrapolorimetry measure-ments Figure 1 shows the superimposed far-UV CD curves of wild-type SNase and G substitute mutants All mutants other than E129G had similar spectra to the wild-type protein with two separated negative peaks at 208 nm and 222 nm, consistent with the pres-ence of an a-helix [13,14] In contrast, the E129G mutant exhibited less CD than the others Thus, while most of the mutations of negatively charged glutamic acid or aspartic acid residues to uncharged glycine resi-dues do not disrupt the secondary structure of SNase, replacement of E129 with G may cause changes in pro-tein secondary structure However, this replacement of
G at E129 does not significantly influence its whole protein stability (see Results of thermal analysis sec-tion below)
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 surrounding hydrophobic environment
of the tryptophan and thus indicate a change in the overall (tertiary) structure of the protein [15] Figure 2
Fig 1 CD spectra of wild-type and SNase mutants CD spectra (far UV) of nine proteins (WT, E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G) All spectra are similar except the E129G spectrum (bold line) Protein concentration, 0.5 mgÆmL)1.
Trang 3shows the fluorescence spectra of the wild-type and
glycine mutants Peak fluorescence intensities of the
mutants at W140 were 21% to 45% lower than that of
the wild-type protein As shown in Fig 2, the peak
amplitude for the E129G mutant was exceptionally
low, with a 45% decrease in fluorescent intensity
relat-ive to the wild-type protein The fluorescence changes
of W140 were in agreement with the thermodynamic
results obtained from thermal unfolding (DHcal) (for
data see the following section and Table 1) These data
indicate that the mutants are much less stable than the
wild-type protein In the E129G mutant, the
trypto-phan at residue 140 may be exposed to the water
environment due to the mutant’s loosened tertiary
structure For those mutants exhibiting only small
decreases in fluorescence intensity, those amino acids
around the tryptophan are probably still in the form
of their native conformation This hypothesis is
sup-ported by the CD results, showing that some
secon-dary structure still exists in these mutants (Fig 1)
Thermal analysis of protein unfolding
Thermal denaturation determined by DSC was used to
measure the stability of the mutant proteins Based on
the measurements of heat capacity over a range of temperatures (DSC curves are shown in Fig 3), parameters such as melting point (Tm), calorimetric enthalpy (DHcal), difference of DHcal of mutant from wild-type (%), heat capacity (DCp) and difference of
DCp of mutant from wild-type (%) were determined and the results for the wild-type protein and 20 mutants are given in Table 1 The DHcaland Tmof the wild-type protein were in good agreement with previ-ous studies [7] The variprevi-ous mutations, however, exis-ted difference in their effects on the stability of SNase Among them, eight mutants (E73G, E75G, E101G, E122G, E129G, D77G, D83G and D95G) showed decreases in DHcalof greater than 20% compared with the wild-type protein, indicating that these charged residues have a strong influence on the unfolding of SNase The heat denaturation curves for the wild-type protein and eight mutants are shown in Fig 3, demon-strating that the mutants have heat denaturation pro-perties distinct from the wild-type protein These mutants had lower DHcal and Tm values, and their DSC curves were shifted toward lower temperatures than the wild-type protein D77G, D83G and D95G mutants, in which aspartic acid residues were replaced
by glycine, had Tmvalues of about 44.14C, 37.21 C and 37.38C, respectively, compared with the wild-type Tm of 50.98C As listed in Table 1, the DHcal values for D77G, D83G and D95G were about 64.35 kcalÆmol)1, 57.50 kcalÆmol)1 and 64.35 kcalÆ mol)1, respectively, 26% to 33% lower than that of the wild-type protein
The thermodynamic parameters of the glycine susti-tution mutants also differed greatly from the wild-type protein The Tm values of these mutants ranged from
35C to 55 C Among them, the E75G and E129G mutants showed the largest decreases in thermal dynamic quantities The Tm values of E75G (37.0C) and E129G (34.39C) were much lower than that of wild-type protein (50.98 C) E129G had the lowest
DHcal of the mutants (47.6 kcalÆmol)1) and E75G had the second lowest (48.2 kcalÆmol)1) TheseDHcalvalues are more than 44.13% (E129G) and 43.43% (E75G) lower than that of the wild-type protein, suggesting that Glu residues at positions 75 and 129 play key roles in maintaining the native structure of the protein Furthermore, two mutants, E75Q and E129Q, were created for consideration The results of thermal dena-turation show that E75Q (DHcal¼ 51.4 ± 2.0 kcalÆ mol)1) and E129Q (DHcal¼ 54.2 ± 0.8 kcalÆmol)1) have approximately more than 40% (vs 43% of E75G) and 37% (vs 44% of E129G), respectively, lower DHcal values than the wild-type protein These outcomes indicate that little influence is exerted by Gly
Fig 2 Steady-state fluorescent spectra of wild-type and mutants
of SNase Spectra of nine proteins (WT, E73G, E75G, E101G,
E122G, E129G, D77G, D83G and D95G) The spectrum of E129G
(bold line) is much lower in intensity than the others Protein
con-centration, 0.4 mgÆmL)1.
Trang 4residue substitution on the destabilization of secondary
structure that leads to the large perturbation of the
stability of the protein tertiary structure (although
there is about 7% difference of thermal energy change between E129Q and E129G)
Discussion
Protein folding and unfolding are driven by the free energy of stabilization when a peptide chain in ran-dom configuration (denatured state, D) folds into the compact three-dimensional structure of the native state (N) [16,17] For SNase, we previously reported the conversion of these two states by the kinetic scheme [18–22] N ¢ D1¢ D2¢ D3, where Di denotes the denatured states of protein and the conversion energy between each state less than 5 kcalÆmol [1] Therefore, the measured energy of thermal unfolding mainly contributes to the conversion of N to D For wild-type SNase, the unfolding energy needed is about 85.9 kcalÆmol)1 (Table 1) This energy is used
to destroy all network forces in the native protein Some significant local forces play roles to help this network frame [7] resist unfolding In the scheme above, the structure and dynamics of the N state of SNase has been elucidated, but relatively little is known about the D state [23] Zhou used the Gaus-sian chain model [24] to point out that an unfolded protein could be viewed as a collection of peptide
Table 1 Comparison of thermodynamic parameters for wild-type SNase and 20 SNase mutants in which negative charged D and E residues were mutated to G Phosphate buffer (25 m M Na2HPO4, 25 m M NaH2PO4, 100 m M NaCl, pH adjusted to 7.0) was used All proteins were used at a concentration of 2 mgÆmL)1 WT, wild-type Difference of DH from WT (%) is calculated by [(DH mutant –DH WT ) ⁄ DH WT] ] · 100 Differ-ence of DC p from WT (%) is calculated by[(DC pmutant –DC pWT ) ⁄ DC p (WT) · 100.
Average Tm
(C) DH (kcalÆmol)1)
Difference of
DH from WT (%)
DC p
(kalÆmol)1ÆK)1)
DDC p
(kcalÆmol)1ÆK)1)
Difference of DC p
from WT (%)
Fig 3 Calometric melting curves of wild-type and mutants of
SNase DSC curves of nine proteins (WT, E73G, E75G, E101G,
E122G, E129G, D77G, D83G and D95G) Lines 3 and 6 represent
E75G and E129G, respectively; the curves are lower in intensity
than the others Protein concentration, 2 mgÆmL)1 Thermodynamic
parameters such as DH cal were calculated based on the description
of Privalov and Potekhin [6].
Trang 5fragments that dynamically vary their conformation
and relative distances For SNase pH denaturation,
the Distates (scheme shown above) can be considered
as many different stages of an assembly with local
structural elements capturing each other However,
we do not favour the author’s theoretical conclusion
[24] that residual electrostatic effects on protein
re-folding are not important due to the nonrandom
interactions between charged amino acids
Experi-mentally, our results based on thermal unfolding and
pH titration [22] indicate that the local electrostatic
interactions at specific positions (E75 and E129,
Table 1) play important roles in stabilizing protein
structure [25,26] Fink and his coworkers [27] have
demonstrated that SNase is only marginally stable at
pH 7 due to a combination of high net protein
charge and low hydrophobicity Anderson et al used
NMR as a tool to measure protein apparent pKa and
emphasized that the electrostatic contribution of each
ionizable group may play a role in the stability of the
folded SNase [28] Schwehm et al also reported that
electrostatic interactions are the most important net
stabilizing factor in SNase for single site mutations
that reverse or neutralize a surface charge [29] On
the contrary, Shortle and coworkers [30] argued that
ionizable amino acids do not contribute greatly to the
stability of SNase Their viewpoint was based on
the similar calculated free energy differences, DDG
(DGmut,H2O–DGwt,H2O) and fluorescence titration
melt-ing points, mGuHCl, of total 104 mutants It should
be noted that Shortle et al used indirect
measure-ments and much theoretical calculation to determine
protein stability, which may account for their
hypo-thesis that ionizalbe residues do not significantly
affect SNase stability Our study on 20 mutants using
thermal analysis is more accurate and we believe this
refects the acutal stability shifts due to single charged
amino acid mutations In our pH titration study
(pH 7.0–2.0), we found that SNase protein could be
denatured by the addition of only 2.5 protons (data
not shown) Therefore, we hypothesize that pH
unfolding SNase is mainly processed by the addition
of 2.5 protons and both E75 and E129 are the targets
of protonation
It is of interest to determine which other amino
acids these glutamic acid residues at positions 75 and
129 might be interacting with to stabilize SNase
struc-ture Based on the X-ray structure (1EYD) [31] of
SNase, the positively charged amino acids expected to
have the most significant interactions with E75
(OE1⁄ OE2)– are H121 (NE2)+ (¼ 3.06 A˚) and K9
(Nz)+(¼ 5.25 A˚) Similarly, the amino acids expected
to have the strongest interactions with E129
(OE1⁄ OE2)– are K110 (Nz)+ (¼ 6.29 A˚) and K133 (Nz)+ (¼ 6.36 A˚) These two regions with predicted strong local interactions are circled in Fig 4 Within the circled areas, other charged amino acids such as D77, D83 and E101 may provide supportive forces to the areas surrounding the interactions with E75 and E129 Privalov and coworkers reported the presence of noncovalent bonding, including an electrostatic bond between E75 and H121, and a hydrogen bond between D77 and T1208 Thus interactions between D77 and E75 and surrounding positively charged amino acids may result in the cooperative formation of two sub-domains: a C terminus comprised of an a-helix and a b-barrel This hypothesis is supported by our thermal DSC data, indicating decreases of 25.09% (D77G) to 43.43% (E75G) in the enthalpic energy of these mutants compared to wild-type SNase Furthermore, D83G, which results in a 33.06% decrease inDH com-pared to wild-type SNase, is also located between the two subdomains These results imply that E75, D77 and D83 are part of the same local stable area From this study, E129G and E101G mutations resulted in decreases of 44.13% and 28.17%, respectively, in ther-mal stability as compared with wild-type protein These residues are located in two adjacent helices of SNase, namely helix 2 (V99 to Q106) and helix 3 (E122 to K134) This finding implies that E129 and E101 form another local stable area through inter-actions with K110 and K133 (Fig 4) and is in agree-ment with the fluorescence measureagree-ments in which the
Fig 4 Local stable segments in SNase The X-ray crystal structure
of wild-type SNase was obtained from the Protein Data Bank The circled areas show local stable segments: interactions of G75 with H121 and K9, and of G129 with K110 and K133 Other charged amino acids such as D73, D83 and E101 reinforce the interactions
of E75 and E129.
Trang 6amplitude of E129G was decreased by about 44%
compared with the wild-type protein
Conclusions
Comparisons of thermal stability and protein
unfold-ing for wild-type SNase and 20 glycine mutants suggest
that the negatively charged aspartic acid and glutamic
acid residues in the protein play unequal roles in
main-taining the protein’s native structure More than one
local stable segment is present: E75 with K9 and H121
form one local stable area and E129 with K110 and
K133, located in two adjacent a-helices (helix 3 and
helix 2, respectively) form another in SNase These
two areas must be significantly perturbed before the
protein can be unfolded
Experimental procedures
Materials
Luria–Bertani (LB) broth and isopropyl thio-b-d-galactoside
were from USB (Cleveland, OH) Salmon testes DNA and
some analytical grade chemicals such as EDTA, Tris⁄ HCl,
CaCl2, NaCl and mineral oil were from Sigma (St Louis,
MO, USA) Salmon testes DNA applied for the enzyme
activity test was used without further purification
Guani-dine hydrochloride and dNTPs were from Boehringer
(Mannheim, Germany) Ethanol (> 99%) was from
Pan-reac (Barcelona, Spain) Urea was a product of Acros,
USA The Stratagene QuickchangeTM kit containing Pfu
DNA polymerase, 10· reaction buffer and DpnI restriction
enzyme was 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, Johns Hopkins University, Baltimore, MD,
USA) was cloned into pTrc-99 A and used to transform
Escherichia coli strain JM105 Plasmid DNA was purified
by the alkaline lysis method (Gibco-BRL, Gaithersburg,
MD, USA; GFXTMkit), and stored at)20 C before being
subject to mutagenesis Twenty complementary 33-mer
oligonucleotides were synthesized to each introduce one
gly-cine at positions 10, 19, 21, 40, 43, 52, 57, 67, 73, 75, 77,
83, 95, 101, 122, 129, 135, 142, 143 and 146 of wild-type
SNase (Life Technologies, Rockville, MD, USA) Similarly,
two complementary 33-mer oligonucleotides were also
syn-thesized to each introduce one glutamine at positions 75
and 129 of the SNase For site-directed mutagenesis, a 10·
reaction buffer (Stratagene, QuickChangeTMkit) was mixed
with 1.5 lL double-stranded DNA template, 1.2 lL of a
pair of complementary oligonucleotides, 1 lL 10 mm each
dNTP and double-distilled H2O to a final volume at 50 lL One microlitre Pfu DNA polymerase (2.5 UÆlL)1) was added to the solution, and the mixture was overlaid with
30 lL mineral oil A PCR consisting of 16 cycles of 50C (1.5 min), 68C (14 min), and 94 C (1 min) was per-formed using a PerkinElmer 480 thermal cycler (Foster City, CA, USA) The wild-type DNA template was then digested by adding 1 lL of DpnI restriction enzyme (10 UÆlL)1) to the PCR mixture and incubating at 37C for 1 h Ten microlitres of the reaction (containing undi-gested mutant plasmid) were used to transform 100 lL of competent JM105 cells The mixture was incubated on ice for 1 h and at 42C for 2 min, followed by a further 2 min incubation on ice After transformation, 800 lL of LB medium were added and incubated was at 37C for 1 h Transformed cells were selected on plates containing ampi-cillin (100 lgÆmL) and mutant DNA was isolated from the resulting colonies Mutant plasmids were identified by BamHI and NcoI restriction digestion and sequences were confirmed by DNA sequencing
DNA sequencing
Plasmid DNA was isolated using a GFXTMMicro Plasmid Prep Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the resulting double-stranded DNA was mixed with 8 lL of BigDyeTMmaster mix (BigDyeTMTerminator Ready Reaction Kit, Applied Biosystems) and 3.2 pmol sequencing primer The final solution was mixed with de-ionized water to a final volume of 20 lL in a 0.5-mL thin-walled PCR tube and overlaid with 40 lL of light mineral oil DNA sequencing was performed by cycle sequencing using 25 cycles of 96C for 30 s, 50 C for 15 s, and 60 C for 4 min in a Perkin-Elmer 480 thermal cycler The exten-sion products were purified by Centri-SepTM spin column chromatography (Princeton Separation, Adelphia, NJ, USA) to remove unincorporated dye terminators Five microlitres of template suppression reagent (PE Applied Biosystems) was mixed with the purified extension prod-ucts The samples were heated at 95C for 2 min and then chilled on ice Capillary electrophoresis was performed using an ABI PRISM 310 Genetic 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 to that of wide-type and were confirmed to have the correct mutant sequence
Protein purification
Escherichia coliJM105 carrying recombinant plasmids were grown in LB broth containing 100 lgÆmL)1 ampicillin at
37C Protein expression was induced by adding isopropyl thio-b-d-galactoside The cells were harvested after 4 h of incubation and suspended in chilled buffer A (6 m urea,
Trang 70.05 m Tris⁄ HCl, 0.2 m NaCl, pH 9.2, filtered through a
0.45 lm membrane) Proteins were collected after two
alco-hol precipitations and stored in buffer B (6 m urea, 0.05 m
Tris⁄ HCl, 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
col-umn) The proteins were dialysed after purification for
2 days at 4C against phosphate buffer (0.025 m
NaH2PO4, 0.025 m NaHPO4, 0.1 m NaCl, pH adjusted to
7.0) and were then lyophilized The average yields of the
recombinant proteins were about 30Æmg L)1 SNase purity
was investigated by SDS⁄ PAGE, gels were stained with
Coomassie blue and analysed by a densitometer, revealing
protein purity of greater than 90% Protein concentration
was determined by measuring the extinction coefficient with
Gill and von Hippel’s method [32]
Circular dichroism
Circular dichroism was performed on wild-type protein
and mutants using a Jasco Model J-720
spectropolarime-ter The spectra were measured between 200 and 320 nm
Wild-type and mutant proteins were dissolved in
phos-phate buffer (0.025 m NaH2PO4, 0.025 m NaHPO4, 0.1 m
NaCl, pH adjusted to 7.0) at a concentration of
0.5 mgÆmL)1 Spectra 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 0.4 mgÆmL)1 Excitation was set at 298 nm and
emis-sions were observed at 350 nm The fluorescence spectra
were measured between 300 and 550 nm with a scanning
speed of 150 nmÆs)1and an excitation slit of 5.0 nm
Calorimetric measurements
Thermal analysis of protein denaturation was performed
by nano DSC (Model 6100 Nano II; Calorimetry Sciences
Corp., Provo, UT, USA) Lyophilized wild-type and
mutant SNase were dissolved in phosphate buffer (25 mm
NaH2PO4, 25 mm NaHPO4, 100 mm NaCl, pH adjusted to
7.0) at a concentration of 2 mgÆmL)1 Samples were
soni-cated for 15 min, and then 1 mL of 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 20C to 75 C under 3 atm at a heating
rate of 1CÆmin)1 The melting point (Tm) was obtained
directly from the DSC curve The enthalpy change (DHcal)
was calculated by the integration of the curve covering area
(Tm was taken as the curve peak point) using origin soft-ware
Acknowledgements
This work wass partially supported by a grant (NSC-92-2311-B-001) from National Science Council, Tai-wan, R.O.C and the theme project of Academia Sinica, Taipei, Taiwan, R.O.C
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