Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant C4S⁄ C18S ⁄ C29S ⁄ C60S in which all Cys residue
Trang 1Shunsuke Kawamura1, Mari Ohkuma1, Yuki Chijiiwa1, Daiki Kohno1, Hiroyuki Nakagawa1, Hideki Hirakawa2, Satoru Kuhara2,3and Takao Torikata1
1 Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, Japan
2 Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan
3 Graduate School of Genetic Resource Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan
Lysozyme, one of the best characterized
carbohydro-lases, cleaves the glycosidic linkage between
N-acetyl-glucosamine (GlcNAc) and N-acetylmuramic acid in
bacterial cell walls This enzyme is classified into
six types, chicken type (C-type) [1–3], phage type
(T4-type) [4,5], goose type (G-type) [6–8], invertebrate
type [9–11], bacteria type [12], and plant type [13], on
the basis of the similarity in amino acid sequences
These different classes of lysozymes have overall
simi-larities in tertiary structure [7,14–17], although their
amino acid sequences are almost entirely different
Much information on the structural properties and enzymatic mechanisms of C-type and T4-type lyso-zymes has been accumulated thus far In particular, hen egg-white lysozyme and human lysozyme, which belong to a class of C-type lysozymes with four disul-fide bonds, and T4 phage lysozyme with no disuldisul-fide bonds, have been intensively studied as model proteins for elucidating enzymatic function and protein stabil-ity In contrast to C-type and T4-type lysozymes, information on G-type lysozyme is limited In verte-brates, the primary structure has been reported for five
Keywords
disulfide bonds; goose-type lysozyme;
ostrich; site-directed mutagenesis; structural
stability
Correspondence
S Kawamura, Department of Bioscience,
School of Agriculture, Tokai University, Aso,
Kumamoto 869-1404, Japan
Fax: +81 967 67 3960
Tel: +81 967 67 3918
E-mail: kawamura@agri.u-tokai.ac.jp
(Received 15 November 2007, revised 3
March 2008, accepted 25 March 2008)
doi:10.1111/j.1742-4658.2008.06422.x
The role of the two disulfide bonds (Cys4–Cys60 and Cys18–Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state The effects of deleting each disulfide bond on protein stability were found to be approxi-mately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S⁄ C18S ⁄ C29S ⁄ C60S) in which all Cys residues were replaced by Ser Moreover, the optimum tem-perature of the catalytic activity for the Cys-free mutant was downshifted
by about 20C as compared with that of the wild-type These results indi-cate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme
Abbreviations
(GlcNAc)n, b-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n; C-type, chicken type; GEL, goose egg-white lysozyme; GlcNAc, N-acetylglucosamine; G-type, goose type; MD, molecular dynamics; OEL, ostrich egg-white lysozyme; T4-type, phage type; b-ME, b-mercaptoethanol.
Trang 2G-type lysozymes, i.e those from ostrich [18], black
swan [19], embden goose [6], cassowary [20], and rhea
[21], and five from chicken [22], flounder [23], carp
[24], salmon [25], and orange-spotted grouper [26]
Additionally, Irwin & Gong [27] reported that
mam-mals and zebrafish carry two G-type lysozyme genes
Recently, invertebrate G-type lysozyme genes and⁄ or
enzyme activity from scallop [28,29] and tunicate [30]
were also reported G-type lysozyme differs from the
C-type in that it is much more specific for
peptide-substituted substrate [31] C-type lysozyme hydrolyzes
a homopolymer (chitin) effectively, whereas G-type
lysozyme is a poor catalyst of the hydrolysis of this
substrate The differences in substrate specificity
between these lysozymes and the mechanistic details of
the catalytic reaction of G-type lysozyme remain
unclear Previously, Honda & Fukamizo reported the
mode of binding of GlcNAc oligomer to goose
egg-white lysozyme (GEL), and postulated that GEL has
six substrate-binding subsites (sites B–G) [32] This
subsite structure was partly visualized in terms of the
crystal structure of the GEL–(GlcNAc)3 complex [14];
however, part of the subsite structure (sites E–G)
remains unknown
On the basis of sequence comparison of G-type
lyso-zymes, we have shown that the amino acid sequences
of three a-helices (a5, a7, and a8) are highly conserved
in this enzyme group [20,21] These three a-helices are
located at the center of the protein molecule, and form
a hydrophobic core in the overall structure of G-type
lysozyme (Fig 1) Recently, using ostrich egg-white
lysozyme (OEL) as a model, we demonstrated the
involvement of Glu73 on a5 (Ala64–Glu73) as a
criti-cal catalytic residue and also indicated the crucial role
of Glu73 in the structural stability of G-type lysozyme,
probably through the interhelical hydrogen bond with Tyr169 on a8 (Tyr169–Gln182) [33] These observa-tions suggest that the core elements (a5, a7, and a8) play an important role in the maintenance of the three-dimensional structure of G-type lysozyme
In addition to the three a-helices, the Cys4–Cys60 and Cys18–Cys29 disulfide bonds (numbering from bird sequences), which are located in the N-terminal region, are completely conserved in avian and mamma-lian G-type lysozymes, although another three Cys res-idues are conserved from mouse to human [20,21,27]
In the crystal structure of GEL (Fig 1), which shares 83% amino acid identity with OEL, these two disulfide bonds are located on the molecular surface, although Cys60 is partially buried in the interior of the protein
It has been shown that the integrity of the native three-dimensional structure of many proteins is pro-moted by the presence of disulfide bonds, because removal of one or more of these linkages results in a reduction in the stability of the native state relative to the denatured state [34–44] On the other hand, none
of the four disulfide bonds was reported to be impor-tant in stabilizing the native structure of the Pseudoal-teromonas haloplanktis a-amylase [45] It was also shown that the Cys191–Cys220 disulfide bond, which
is highly conserved in the trypsin family of serine pro-teases, is not essential for the catalytic function, struc-ture and stability of trypsin [46] Therefore, analysis of the role of the disulfide bonds in activity and stability will be useful for our understanding of the structure– function relationship of G-type lysozyme The present article describes a site-directed mutational analysis of OEL to address the role of the disulfide bonds in G-type lysozyme
Results and Discussion Choice of residues for mutagenesis
A striking difference within G-type lysozymes is the variation in Cys content There are four conserved Cys residues in avian and mammalian G-type lysozymes, which form two intramolecular disulfide bonds in the mature proteins [20,21,27] The crystal structure of GEL shows that the Cys18–Cys29 disulfide bond con-nects the N-terminus of a-helix 1 with a loop between a-helices 1 and 2, and the Cys4–Cys60 disulfide bond connects the N-terminal long loop with a loop between a-helices 4 and 5 The G-type lysozymes found in fish have either no Cys residue, as in flounder and grouper [23,26], one, as in carp and salmon [24,25], or two (no potential to form an intramolecular disulfide bond),
as in zebrafish [27] The absence of intramolecular
Fig 1 The three-dimensional structure of GEL The structure was
created using the coordinate file, Protein Data Bank entry 153L
[14] The two disulfide bonds (Cys4–Cys60 and Cys18–Cys29) and
three a-helices (a5, a7, and a8) are shown in blue and green,
respectively The side chain of Glu73 is also shown in red The
figure was generated using MOLSCRIPT (v 2.1.2).
Trang 3disulfide bonds seems to be a common characteristic
among the fish G-type lysozymes The G-type
lyso-zymes in invertebrates have six to 13 Cys residues
other than the four Cys residues conserved in avian
and mammalian G-type lysozymes, which renders the
disulfide patterns of invertebrate G-type lysozymes
quite different from those of the bird and mammalian
lysozymes [28–30] As the locations of disulfide bonds
in invertebrate G-type lysozymes have not yet been
identified, we decided to focus our attention on the
Cys18–Cys29 and Cys4–Cys60 disulfide bonds, which
are absolutely conserved in avian and mammalian
G-type lysozymes
Expression and characterization of mutant
proteins
To investigate the contribution of the two disulfide
bonds to the activity and stability of G-type lysozyme,
three mutant proteins (C4S⁄ C60S, C18S ⁄ C29S, and
C4S⁄ C18S ⁄ C29S ⁄ C60S), in which each of the disulfide
bonds was singly or together disrupted by Cysfi Ser
mutations, were initially constructed The Ser residue
was chosen because it is structurally similar to Cys
except that it contains a hydroxyl group instead of a
thiol group The mutant proteins were expressed and
purified in the same manner as used for the wild-type
[33] The yields of the mutant proteins were
com-parable to that of the wild-type, approximately
60–70 mgÆL)1 The purified proteins were found to be
homogeneous on analysis by SDS⁄ PAGE, and gave a
single peak on RP-HPLC (data not shown) The
N-ter-minal sequence of each mutant protein was determined
to be Ser-Arg-Thr-Gly, which coincided with that of
the wild-type, indicating that each mutant was correctly
processed at the C-terminus of the a-factor signal The
integrity of the mutant proteins was confirmed by
mea-surements of far-ultraviolet CD The CD spectra of the
three mutants were almost indistinguishable from that
of the wild-type (Fig 2), indicating that the backbone
conformation of the mutant proteins is practically the
same as that of the wild-type Thus, it appears that
none of the disulfide bonds are critically important to
the folding process of G-type lysozyme
Effects of mutations on catalytic activity
We previously reported that the recombinant OEL
preferentially hydrolyzes the third glycosidic linkage
from the nonreducing end of (GlcNAc)6, and the
cleavage pattern seen for (GlcNAc)5 is similar to that
seen for (GlcNAc)6 [47] To examine the effects of the
mutations on the catalytic activity of G-type lysozyme,
we initially analyzed the activities of the wild-type and its mutants by monitoring the enzyme-catalyzed lysis
of Micrococcus luteus cells, which is a high molecular mass polymeric substrate with a highly negative charge Mutant C4S⁄ C60S had lytic activity to the same extent as the wild-type, exhibiting 99.0% activity The lytic activities of mutants C18S⁄ C29S and C4S⁄ C18S ⁄ C29S ⁄ C60S were 76.5% and 70.6% of that
of the wild-type, respectively (data not shown) As the substrate used for lytic activity is chemically heteroge-neous, the activities of the wild-type and three mutant proteins were more precisely evaluated by measuring the enzyme-catalyzed hydrolysis of (GlcNAc)5 (Fig 3) Consistent with the results obtained in the M luteus assay, the wild-type and mutant C4S⁄ C60S hydrolyzed the initial substrate (GlcNAc)5 almost completely after
240 min of reaction, and (GlcNAc)5 was hydrolyzed mainly to (GlcNAc)2+ (GlcNAc)3 with much less cleavage to (GlcNAc)1+ (GlcNAc)4 Mutants C18S⁄ C29S and C4S ⁄ C18S ⁄ C29S ⁄ C60S hydrolyzed (GlcNAc)5 to produce (GlcNAc)2 and (GlcNAc)3, as
in the case of the wild-type, although the overall rates
of hydrolysis were slightly affected by each of the mutations: the two mutants took 420 min to hydrolyze most of the (GlcNAc)5 These results are consistent with the fact that each of the two disulfide bonds is
–30 –20 –10 0 10 20 30 40 50 60
190 210 230 250
Wavelength (nm)
3 (deg cm
2 ·dmol
Wild type C18S/C29S C4S/C60S C4S/C18S/C29S/C60S
Fig 2 CD spectra of the wild-type and its three mutant proteins in the far-UV region.
Trang 4far apart from the catalytic glutamate, Glu73, in the
crystal structure of GEL (even the nearest two
resi-dues, Cys18 and Glu73, are located 16.8 A˚ apart from
each other) Although the reason for the slight
reduc-tion in the activity of the two mutants is presently
obscure, none of the disulfide bonds is considered to
be critically important to the catalytic function of
G-type lysozyme This observation, together with the
result obtained by CD analysis, implies that G-type
lysozyme can fold and function in the absence of both
disulfide bonds This is consistent with the findings
that the flounder and salmon G-type lysozymes, which
have no disulfide bonds in their native forms, and the
scallop G-type lysozyme, which has six Cys residues
other than the well-conserved four Cys residues in
birds, possessed lytic activity against Micrococcus
lysodeikticus[23,25,29]
Effects of mutations on stability
Disulfide bonds have been suggested to play an
impor-tant role in maintaining structural integrity and protein
stabilization This conclusion has been supported by
characterization of mutants of various proteins in
which disulfide bonds have been either deleted or
mod-ified For example, Pace et al [34] reported that
dis-ruption of one and two disulfide bonds in
ribonuclease T1 caused decreases in conformational
stability by 3.4 and 7.2–9.3 kcalÆmol)1, respectively
Many other examples can be found in the reviews by
Wetzel [48] and Bets [49] It is widely accepted that the
stabilizing effect of a disulfide bond can be attributed
principally to the destabilization of the unfolded form
by the loss of conformational entropy imposed by the
crosslink [34,36,39,50–54] However, the mechanism of protein stabilization by disulfide bond formation is difficult to resolve, because the disulfide bond may influence the enthalpy and entropy of both the native and unfolded states of the protein [49]
The thermal unfolding of the wild-type protein has been investigated using CD and fluorescence spectros-copy, and led to the observation that the unfolding transition of the wild-type is well represented by a two-state mechanism at pH 5.0 in the presence of 0.5 m guanidine hydrochloride [33] Like the wild-type pro-tein, the three mutants (C18S⁄ C29S, C4S ⁄ C60S, and C4S⁄ C18S ⁄ C29S ⁄ C60S) reversibly unfolded in a single cooperative fashion under these conditions The ther-mal unfolding curves of the wild-type and three mutant proteins obtained with fluorescence measurements are shown in Fig 4A Replacing Cys18 and Cys29 or Cys4 and Cys60 with a pair of Ser residues had significant effects on the thermal unfolding of the mutant proteins The Tmvalues for mutants C18S⁄ C29S and C4S ⁄ C60S were decreased by 6.3C and 9.5 C, respectively, as compared to 60.6C for the wild-type (Table 1) The Cys mutations reduced the thermostability of the pro-teins by 3.11 and 4.29 kcalÆmol)1 for mutants C18S⁄ C29S and C4S ⁄ C60S, respectively, at 60.6 C The combination of these destabilizing mutations (C4S⁄ C18S ⁄ C29S ⁄ C60S) caused a further decrease in thermostability relative to the wild-type by 15.3C (DDG:)6.14 kcalÆmol)1) (Table 1)
The contribution of the disulfide bonds to the struc-tural stability of OEL was further assessed by means
of unfolding experiments with guanidine hydrochloride
as a denaturant Figure 4B shows the guanidine hydro-chloride-induced unfolding curves of the wild-type and
Fig 3 Time course plots of (GlcNAc)5
degradation by the wild-type and its three
mutant proteins The enzymatic reaction
was performed in 10 m M sodium acetate
buffer (pH 4.0) at 40 C Numerals in the
figures are the polymerization degrees of
the reaction product species Relative error
indicates the recovery of the observed value
at each reaction time calculated as
described in Experimental procedures The
solid lines were drawn by roughly following
the experimental data points.
Trang 5mutant proteins obtained with fluorescence
measure-ments The transitions of the three mutant proteins
were highly cooperative The unfolding transitions of
the mutant proteins occurred at lower concentrations
of guanidine hydrochloride than that of the wild-type:
the Cmvalues were reduced, as compared with that of the wild-type, by 0.47, 0.70 and 1.07 m for mutants C18S⁄ C29S, C4S ⁄ C60S, and C4S ⁄ C18S ⁄ C29S ⁄ C60S, respectively (Table 2) The DGH2O values of unfolding indicated that the three mutants, C18S⁄ C29S, C4S⁄ C60S, and C4S ⁄ C18S ⁄ C29S ⁄ C60S, were destabi-lized by 2.33, 3.33 and 4.86 kcalÆmol)1, respectively, at
0 m guanidine hydrochloride in comparison to the wild-type
The decrease in the stability of the least stable C4S⁄ C18S ⁄ C29S ⁄ C60S mutant was further confirmed
by CD measurements: its thermal and guanidine hydrochloride transition curves derived from the CD and fluorescence data completely coincided, and the
Tm and Cm values obtained with CD were in good agreement with the data determined with fluorescence (Fig 5, and Tables 1 and 2) The coinciding transitions derived from these two different methods also indicate that the Cys-free mutant undergoes thermal and guani-dine hydrochloride-induced denaturation, which is con-sidered to be a reversible two-state process as observed
in the wild-type It is thus likely that the fold-ing⁄ unfolding pathway of G-type lysozyme does not significantly change in the absence of one or both of the disulfide bonds, which supports the notion that the presence of a complete set of disulfide bonds is not required for the folding process of G-type lysozyme
To corroborate the importance of the disulfide bonds in the structural stability of OEL, two mutant proteins (C18A⁄ C29A and C4A ⁄ C60A), in which Cys18 and Cys29 or Cys4 and Cys60 were replaced
by Ala, respectively, were constructed and analyzed with respect to their guanidine hydrochloride dena-turation (Fig 4B and Table 2) It was found that mutants C18A⁄ C29A and C4A ⁄ C60A exhibited the same stabilities as mutants C18S⁄ C29S and C4S⁄ C60S, respectively, which strongly suggests that the observed destabilization arising from the Cys to Ser or Ala mutations is not due to negative side
–0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
Temperature (ºC)
Wild-type C18S/C29S C4S/C60S C4S/C18S/C29S/C60S Wild-type (reduced)
–0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
GdnHCl ( M )
Wild-type C18S/C29S C4S/C60S C4S/C18S/C29S/C60S C18A/C29A C4A/C60A
A
B
Fig 4 Thermal and guanidine hydrochloride (GdnHCl)-induced
unfolding curves of the wild-type and its mutant proteins obtained
by fluorescence measurements (A) and (B) show the thermal and
guanidine hydrochloride unfolding curves of the wild-type and its
mutant proteins, respectively Experimental details are described in
Experimental procedures The thermal unfolding curve of the
wild-type treated with 0.1 M b-ME for reduction is indicated as
‘wild-type (reduced)’.
Table 1 Parameters characterizing the thermal denaturation of the wild-type and the three mutants Thermodynamic parameters were cal-culated from the thermal unfolding curves presented in Figs 4A and 5A All values are the averages of at least two determinations Data for the wild-type protein were reported by Kawamura et al [33] Errors are within ± 0.3 C for Tm , ± 5.5 kcalÆmol)1for DHm, and ± 0.016 kca-lÆmol)1K for DSmfor the wild-type protein, determined from four independent experiments Flu, fluorescence.
Protein Method DHm(kcalÆmol)1) DSm(kcalÆmol)1ÆK) Tm(C) DTm(C) DDG (kcalÆmol)1)
Trang 6effects of the introduction of the present mutations,
but is due mainly to deletion of the disulfide
bond(s)
We also examined the thermal stability of the wild-type treated with 0.1 m b-mercaptoethanol (b-ME) for reduction and compared it with those of the wild-type and mutant C4S⁄ C18S ⁄ C29S ⁄ C60S (Fig 4A and Table 1) The reduced wild-type exhibited a marked decrease in thermostability relative to the nonreduced wild-type of 14.4C (DDG: )4.85 kcalÆmol)1) The Tm value for the reduced wild-type was 46.2C, a value almost identical to that of the Cys-free mutant (Tm45.3C) No change was observed for the Cys-free mutant when it was melted in the presence of 0.1 m b-ME (data not shown)
In addition, the temperature dependence of the cata-lytic activity against (GlcNAc)5 for the Cys-free mutant was examined, and the result was compared with that for the wild-type (Fig 6) The wild-type protein exhibited the highest activity at 60C, and the activity was drastically reduced at 65C or above In contrast, the optimum temperature for the Cys-free mutant was decreased to 40C, which was about
20C lower than that of the wild-type, and a remark-able drop of the activity was observed above this temperature All of these results indicate that the two disulfide bonds are directly involved in the structural stability of G-type lysozyme Interestingly, the optimum temperature of the lytic activity for the floun-der G-type lysozyme was shown to be 25C by lyso-plate assay [23] Our results suggest that the low optimum temperature observed for the flounder lyso-zyme could be a consequence of the absence of the two intrachain disulfide bonds In the case of inverte-brate G-type lysozymes, the high content of Cys residues suggests their importance in protein stability
We noted that the decrease in the Tmvalue for mutant C4S⁄ C18S ⁄ C29S ⁄ C60S ()15.3 C) agreed well with the sum of the decreases in the Tm values for mutants C4S⁄ C60S and C18S ⁄ C29S ()15.8 C) The DCmvalue for the Cys-free mutant ()1.07 m) was also found to be
Table 2 Parameters characterizing the guanidine hydrochloride denaturation at pH 5.0 and 30 C Parameters were calculated from the gua-nidine hydrochloride unfolding curves presented in Figs 4B and 5B All values are the averages of at least two determinations Data for the wild-type protein were reported by Kawamura et al [33] Errors are within ± 0.03 M for Cm, ± 0.09 kcalÆmol)1Æ M for m, and ± 0.08 kcalÆmol)1 for DG H2O for the wild-type protein, determined from four independent experiments Flu, fluorescence.
Protein Method m (kcalÆmol)1Æ M ) Cm( M ) DCm( M ) DG H2O (kcalÆmol)1) DDG H2O (kcalÆmol)1)
–0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
Temperature (ºC)
Wild-type (Flu) Wild-type (CD) C4S/C18S/C29S/C60S (Flu) C4S/C18S/C29S/C60S (CD)
–0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
GdnHCl( M )
Wild-type (Flu) Wild-type (CD) C4S/C18S/C29S/C60S (Flu) C4S/C18S/C29S/C60S (CD)
A
B
Fig 5 Thermal and guanidine hydrochloride (GdnHCl)-induced
unfolding curves of the wild-type and mutant C4S ⁄ C18S ⁄
C29S ⁄ C60S obtained by CD and fluorescence measurements (A)
and (B) show the thermal and guanidine hydrochloride unfolding
curves, respectively Experimental details are described in
Experi-mental procedures The unfolding curves of the wild-type and
mutant C4S ⁄ C18S ⁄ C29S ⁄ C60S obtained with CD are indicated as
‘wild-type (CD)’ and ‘C4S ⁄ C18S ⁄ C29S ⁄ C60S (CD)’, respectively,
and those of the wild-type and mutant C4S ⁄ C18S ⁄ C29S ⁄ C60S
obtained with fluorescence are indicated as ‘wild-type (Flu)’ and
‘C4S ⁄ C18S ⁄ C29S ⁄ C60S (Flu)’, respectively.
Trang 7nearly equal to the sum of the DCm values for the
corresponding double mutants ()1.17 m) These findings
indicate that the effects on the protein stability of
delet-ing the disulfide bonds are approximately additive As
the locations of the two disulfide bonds are far from
each other in the crystal structure of GEL (Fig 1), the
individual disulfide bonds probably contribute to the
structural stability of G-type lysozyme in an
indepen-dent manner rather than in a cooperative manner
We can assume two mechanisms by which the
disul-fide bonds affect stability One is the entropic effect on
the unfolded forms, and the other is the effect of the
amino acid substitutions on the native forms
Theoreti-cal approaches have suggested that the entropic effect
(DS) will be related to the size of the loop enclosed by
the crosslink (n) A commonly used approximation is
that derived by Pace et al [34]: DS =)2.1–1.5 ·
R· ln n (calÆmol)1ÆK), where R is the gas constant
According to the equation, the increases in entropy in
the unfolded proteins caused by deletion of the Cys18–
Cys29 and Cys4–Cys60 disulfide bonds are 9.51 and
14.15 calÆmol)1ÆK, respectively In terms of free energy,
the expected entropic destabilization ()TDS) of
mutants C18S⁄ C29S and C4S ⁄ C60S is )3.17 and
)4.72 kcalÆmol)1, respectively, at the Tm value of the
wild-type at pH 5.0 (60.6C) These theoretical values are in good agreement with the observed DDG values for mutants C18S⁄ C29S ()3.11 kcalÆmol)1) and C4S⁄ C60S ()4.29 kcalÆmol)1) (Table 1) The sum of the theoretical values for the two double mutants ()7.40 kcalÆmol)1) is also comparable to the observed DDG value for the Cys-free mutant ()6.14 kcalÆmol)1) These results suggest that the increase in entropy of the unfolded state is a dominant factor determining the DDG In the case of the guanidine hydrochloride denaturation, the expected entropic destabilization at
30C is )2.88 and )4.29 kcalÆmol)1 for mutants C18S⁄ C29S and C4S ⁄ C60S, respectively These theo-retical values are close to but lower than the observed DDGH2O values for mutants C18S⁄ C29S ()2.33 kcalÆ mol)1) and C4S⁄ C60S ()3.33 kcalÆmol)1) (Table 2) However, as a small error in m results in a large devia-tion in DGH2O, due to a long extrapolation to 0 m denaturant, and as the concentration of guanidine hydrochloride at the midpoint of the denaturation (Cm) is regarded as the most reliable parameter for estimation of protein stability [55], the destabilization energies (DDGD) of the three mutants (C18S⁄ C29S, C4S⁄ C60S, and C4S ⁄ C18S ⁄ C29S ⁄ C60S) were recalcu-lated using the equation [56] DDGD= m¢(C¢m) Cm), where m¢ and C¢m are the values for the mutant, and
Cmis the value for for the wild-type The destabiliza-tion energies thus obtained are )2.66 and )4.09 kcalÆ mol)1 for mutants C18S⁄ C29S and C4S ⁄ C60S, respec-tively, which are in good agreement with the respective theoretical values The DDGD value for the Cys-free mutant ()6.83 kcalÆmol)1) is also nearly equal to the sum of the theoretical values for the corresponding double mutants ()7.17 kcalÆmol)1) These findings sug-gest that deletion of one or both of the disulfide bonds increases the conformational entropy of the unfolded state, thereby stabilizing the unfolded state and, as a result, destabilizing the protein thermodynamically This is in agreement with the proposal that stabiliza-tion of proteins by disulfide bonds can be essentially ascribed to a decrease of the conformational entropy
of the unfolded state Cooper et al [36] showed that the reduction in Tm resulting from selective disruption and modification of the Cys6–Cys127 disulfide bond of hen egg-white lysozyme is totally attributable to an increase in the entropy difference between the native and denatured states In contrast, Doig & Williams [57] reported, from a thermodynamic analysis on six small proteins, that the dominant effect of disulfide bonds on stability is enthalpic The thermodynamic characterization of a mutant human lysozyme lacking the Cys77–Cys95 disulfide bond showed that the decrease in DGH2O for the mutant protein was caused
Temperature (ºC)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
10 20 30 40 50 60 70 80 90
Wild-type C4S/C18S/C29S/C60S
Fig 6 Temperature dependence of the catalytic activity for the
hydrolysis of (GlcNAc)5 by the wild-type and mutant
C4S ⁄ C18S ⁄ C29S ⁄ C60S The concentrations of hydrolyzed
(Glc-NAc)5 at each temperature were calculated by subtracting the
concentration of the remaining (GlcNAc)5 from that of the initial
(GlcNAc)5substrate.
Trang 8by both entropic and enthalpic factors [37] Therefore,
detailed calorimetric measurements of the mutant
pro-teins will be required to determine the thermodynamic
parameters precisely
Heat inactivation
Next, we examined the stability against irreversible
heat inactivation for the wild-type and mutant
C4S⁄ C18S ⁄ C29S ⁄ C60S Unexpectedly, no difference
was observed in the irreversible heat inactivation of
these enzymes (data not shown) Both enzymes
regained 60% and 20% of their activities after 2 h at
80C and 90 C, respectively This is consistent with
the recent finding that the salmon G-type lysozyme
with no disulfide bonds, which has an optimum
tem-perature of 30C, regained 30% of its activity
immedi-ately after incubation at 90C for 3 h [25] As
suggested by Kyomuhendo et al [25], it appears that
OEL, as in the case of the salmon protein, possesses
an extraordinary capacity to correctly refold its
struc-ture When considered together with the result
obtained for the salmon protein, this indicates that,
probably, no disulfide bonds are required for the
fold-ing process of G-type lysozyme
Homology modeling and MD (molecular
dynamics) simulation
The tertiary structures of the wild-type and mutant
C4S⁄ C18S ⁄ C29S ⁄ C60S were constructed by homology
modeling on the basis of the X-ray structure of GEL [14] The Ramachandran plot provided by procheck ensured very good confidence for the wild-type and the mutant protein The reliabilities of the constructed structures were with 94.3% residues in the most favored regions and 5.7% in additional allowed regions There were no residues in the generously allowed regions and disallowed regions in the two proteins
Figure 7A shows the distribution of the B-factors
of the main chain atoms (N, Ca, C, and O) in the MD-generated average structural models of the wild-type and mutant C4S⁄ C18S ⁄ C29S ⁄ C60S The mole-cules are colored according to the B-factor, from dark blue for low B-factor to red for high B-factor Although the overall structure of the mutant was quite similar to that of the wild-type, an increase in B-factors throughout the molecule was observed in the Cys-free mutant This increase indicates that the conformation of the mutant protein becomes more flexible Figure 7B shows the plots of the B-factors
of the main chain atoms versus each residue for the wild-type and the Cys-free mutant All B-factors of the Cys-free mutant were substantially higher than those of the wild-type The average B-factors of the main chain atoms for the wild-type and the Cys-free mutant in the 200 samples obtained by the MD sim-ulations were 36.4 and 63.1 A˚2, respectively These findings suggest that the two disulfide bonds in G-type lysozyme act to keep the protein molecule folded tightly
A
B
Fig 7 (A) Residue flexibilities calculated
for the wild-type and mutant C4S⁄ C18S ⁄
C29S ⁄ C60S The ribbon diagram was
colored on the basis of the amplitudes of
fluctuations (B-factor) of individual residues.
A blue-to-red color spectrum is used to
represent different levels of flexibilities,
where the smallest motions are in blue and
the highest ones are in red (B) Amplitudes
of B-factors of each amino acid residue The
B-factors for the wild-type and mutant
C4S⁄ C18S ⁄ C29S ⁄ C60S are shown as blue
and pink lines, respectively The locations of
a-helices and b-sheets in the wild-type are
shown as red and sky-blue lines,
respec-tively The locations of the four Cys residues
are shown as green bars.
Trang 9Our results demonstrated experimentally that the
indi-vidual disulfide bonds contribute significantly to the
structural stability of G-type lysozyme in an
indepen-dent manner The results also suggested that the
reduced stabilities caused by deletion of one or both
of the disulfide bonds are due mainly to entropic
effects The MD data showed that deletion of the
two disulfide bonds makes the protein conformation
more flexible It is thus speculated that the formation
of the disulfide bonds is involved in the structural
stability of G-type lysozyme by reducing the
confor-mational entropy of the unfolded state and by
increasing the rigidity of the protein molecule We
also found that deletion of both disulfide bonds does
not prevent the proper folding into the catalytically
active conformation of G-type lysozyme This
sug-gests that the formation of the two disulfide bonds of
G-type lysozyme occurs late in the folding process,
and that these disulfide bonds can be formed
inde-pendently rather than sequentially It is therefore
sug-gested that the structure around the mutation sites,
the N-terminal region in this case (Arg1–Cys60), may
not be responsible for the folding initiation site of
G-type lysozyme When the overall results are taken
into account, the two disulfide bonds of G-type
lyso-zyme may confer stability after the protein reaches its
final folded form in the absence of both disulfide
bonds On the other hand, we have shown that
G-type lysozyme has a structurally invariant core
composed of three a-helices (a5, a7, and a8) [20,21]
Previous investigations of protein folding suggested
that a-helical structures are formed at an early stage
in protein folding [58–63] As G-type lysozyme was
considered to require no disulfide bonds for folding
and function, we suppose that the three a-helices may
pack together in the early stage of the folding process
and act as nucleation sites around which the structure
can be formed
Experimental procedures
Materials
All enzymes used for DNA manipulation were purchased
from TaKaRa (Otsu, Japan) and Toyobo (Osaka, Japan)
The oligonucleotides used were from Hokkaido System
Sci-ence (Sapporo, Japan) Escherichia coli strain JM109 was
used for the transformation and propagation of
recombi-nant plasmids Multi-Copy Pichia Expression kits, including
expression plasmid pPIC9K and host strain GS115, were
obtained from Invitrogen (Carlsbad, CA, USA)
N-Acetyl-glucosamine oligosaccharides [(GlcNAc)n] were prepared by acid hydrolysis of chitin followed by charcoal celite column chromatography [64] M luteus cells were from Sigma (St Louis, MO, USA) Other reagents were of analytical or biochemical grade
Preparation of mutant proteins
Recombinant OEL with an extra Ser at the N-terminus was prepared as described previously [33] and used throughout this study as the wild-type It should be noted that the additional Ser residue at the N-terminus had little effect on the secondary structure, substrate-binding ability, lytic activity and structural stability of OEL [33] A plasmid harboring the wild-type sequence (pGSer–OEL) [33] was used as a template DNA for site-directed mutagenesis The oligonucleotide primers used were 5¢-GCCCTCGAGAAAAGATCTAGAACTGGATCT TACGGAG-3¢ for C4S, 5¢-GCCCTCGAGAAAAGATC TAGAACTGGAGCTTACGGAG-3¢ for C4A, 5¢-CAAA AGCTTTCTGTCGATCCAGC-3¢ for C60S, 5¢-CAAAAG CTTGCTGTCGATCCAGC-3¢ for C60A, 5¢-TCTTCTAA GTCTGCTAAGCCAGAAAAGCTGAACTACTCTGGA GTTG-3¢ for C18S ⁄ C29S, and 5¢-TCTGCTAAGTCTGC TAAGCCAGAAAAGCTGAACTACGCTGGAGTTG-3¢
method [65]) and verified by DNA sequencing Mutants
con-secutive rounds of mutagenesis To create the gene
described for the wild-type [33] The purity was
N-terminal amino acid sequence was determined with a Shimadzu model PPSQ21 sequencer (Shimadzu Co., Kyoto, Japan) The protein concentration was measured
by amino acid analysis with a Hitachi Model L-8500A amino acid analyzer (Hitachi High-Technologies Co., Tokyo, Japan)
CD spectra
spec-tropolarimeter (Japan Spectroscopic Co., Tokyo, Japan)
The data were expressed in terms of mean residue elliptic-ity The path-length of the cells was 0.1 cm for far-UV CD spectra (190–260 nm) Each spectrum was corrected by subtracting the spectrum of the buffer
Trang 10Assay of enzymatic activity
assayed using lyophilized cells of M luteus as a substrate
One hundred microliters of lysozyme (final concentration
0.015 lm) was added to 3 mL of a suspension of M luteus
adjusted to A 0.9 at 540 nm with 0.1 m sodium phosphate
the first 5 min of linear decrease in absorbance at 540 nm
mixture, containing 0.1 mm lysozyme and 1 mm (GlcNAc)5,
was incubated in 10 mm sodium acetate buffer (pH 4.0)
After a given reaction time, 200 lL of the reaction mixture
was withdrawn and rapidly chilled in a KOOL KUP
(Towa, Tokyo, Japan) The reaction mixture was
centri-fuged at 4000 g for 1h with Ultrafree C3LGC (Millipore,
Billerica, MA, USA), and the filtrate was lyophilized The
dried sample was dissolved in 50 lL of ice-cold water,
and then 10 lL of the solution was applied to a TSKgel
in a JASCO 800 series HPLC column Elution was
per-formed with distilled water at room temperature and a flow
concentra-tion was calculated from the peak area monitored as the
UV absorption at 220 nm, using the standard curve
obtained for authentic saccharide solutions The relative
the recovered concentration of all chito-oligosaccarides in
The temperature dependence of the catalytic activity for
in 10 mm sodium acetate buffer (pH 4.0) and incubated at
various temperatures for 5 min Then, the enzyme dissolved
in the same buffer was added, and the activity was
mea-sured at the designated temperature for 30 min (wild-type)
Thermal unfolding
Reversible thermal unfolding was monitored by CD and
fluorescence measurements as described previously [33] CD
measurements at 222 nm were performed with a
Jas-co J-600 spectropolarimeter using a 0.1 cm cuvette The
flu-orescence intensities at 360 nm, excited at 280 nm, were
measured with a Hitachi F-4500 Fluorescence
sodium acetate buffer (pH 5.0) containing 0.5 m guanidine
hydrochloride These conditions were chosen for complete
reversibility of the thermal denaturation [33] In the
solution of the reduced wild-type, 0.1 m b-ME was also added The water-jacketed cell containing each sample was heated for 5 min at a given temperature by a thermostati-cally regulated circulating-water bath All samples were fully equilibrated at each temperature before measurement The temperature of sample solutions was directly measured using a TX1001 thermometer (Yokokawa M&C Co., Tokyo, Japan) To facilitate comparison between the two sets of unfolding curves, the experimental data were normalized as follows The fraction of unfolded protein was calculated from either the CD values or fluorescence intensities by linearly extrapolating the pretransition and post-transition base lines into the transition zone, and then plotted against temperature Assuming that the unfolding equilibrium involves a two-state mechanism, the unfolding curves were subjected to a least squares analysis to deter-mine the midpoint temperatures (Tm) and thermodynamic
are negligible compared to DSm, the difference in the free
between the mutant and wild-type proteins (DDG) was
proteins
Guanidine hydrochloride unfolding
Guanidine hydrochloride-induced unfolding curves were also determined by monitoring two different parameters at
is the intrinsic fluorescence (excitation at 280 nm and
acetate buffer (pH 5.0) with varying concentrations of
fully equilibrated at each denaturant concentration before
under these conditions, and the unfolding data were analyzed on the basis of a two-state model From the guanidine hydrochloride unfolding profiles, the difference
in free energy change between the folded and unfolded states (DG) was calculated according to Pace [55] The free
DG on the guanidine hydrochloride concentration (m) were determined by least squares fitting of the data for the
m[gua-nidine hydrochloride] The guam[gua-nidine hydrochloride con-centration at the midpoint of the transition (DG = 0) was
wild-type and mutant proteins were calculated by subtract-ing the value of the wild-type from those of mutant proteins