Protein Engineering vol.16 no.1 pp.5–9, 2003
DOI: 10.1093/proeng/gzg001
Buried water molecules contribute to the conformational stability
of a protein
Kazufumi Takano1,2, Yuriko Yamagata3 and
Katsuhide Yutani1,4
1Institute
for Protein Research, Osaka University, Yamadaoka, Suita, Osaka
565-0871 and 3Graduate School of Pharmaceutical Sciences, Kumamoto
University, Oe-honmachi, Kumamoto 862-0973, Japan
4Present
address: Graduate School of Sciences, Kwansei Gakuin University,
Gakuen, Sanda, Hyogo 669-1337, Japan
2To
whom correspondence should be addressed. Present address: Graduate
School of Engineering, Osaka University, Yamadaoka, Suita, Osaka
565-0871, Japan.
E-mail: [email protected]
This study sought to attain a better understanding of the
contribution of buried water molecules to protein stability.
The 3SS human lysozyme lacks one disulfide bond between
Cys77 and Cys95 and is significantly destabilized compared
with the wild-type human lysozyme (4SS). We examined
the structure and stability of the I59A-3SS mutant human
lysozyme, in which a cavity is created at the mutation site.
The crystal structure of I59A-3SS indicated that there were
ordered new water molecules in the cavity created. The
stability of I59A-3SS is 5.5 kJ/mol less than that of 3SS.
The decreased stability of I59A-3SS (5.5 kJ/mol) is similar
to that of Ile to Ala mutants with newly introduced water
molecules in other globular proteins (6.3 ⍨ 2.1 kJ/mol),
but is less than that of Ile/Leu to Ala mutants with empty
cavities (13.7 ⍨ 3.1 kJ/mol). This indicates that water
molecules partially compensate for the destabilization by
decreasing hydrophobic and van der Waals interactions.
These results provide further evidence that buried water
molecules contribute to protein stability.
Keywords: cavity/human lysozyme/mutant protein/protein
stability/water molecule
Introduction
Water molecules are frequently present in the interior of
globular proteins (Edsall and McKenzie, 1983; Rashin et al.,
1986; Kossiakoff et al., 1992; Ernst et al., 1995). There is a
question regarding whether a buried water molecule stabilizes
or destabilizes the protein structure. Each buried water molecule
in protein molecules usually forms several hydrogen bonds
with neighboring residues (Hubbard et al., 1994), leading to
favorable van der Waals interactions as a result of the tight
packing of groups in the protein interior (Williams et al.,
1994). These effects have been expected to contribute favorably
to protein stability. However, the hydrogen bonding potential
of a water molecule inside a protein structure has been less
exploited than that in the aqueous phase. Furthermore, there
are entropic costs for localizing a water molecule within a
protein (Dunitz, 1994) and energy costs for hydration of the
cavity (Zhang and Hermans, 1996). These could destabilize
protein structures.
Many theoretical studies have been conducted to understand
© Oxford University Press
the role of buried water molecules in protein stability (Wade
et al., 1991; Madan and Lee, 1994; Fischer and Verma, 1999).
However, there have been only a few experimental studies
because it is difficult to estimate experimentally. We previously
succeeded in estimating the role of buried water molecules by
examining Ile to Ala/Gly mutants of the human lysozyme
(Takano et al., 1997a). The results demonstrated that mutants
with additional water molecules in the created cavity are
destabilized less than mutants of the human lysozyme with
empty cavities, indicating that the buried water molecules
stabilize the protein structure (Takano et al., 1997a). It is
important to verify empirically whether this conclusion is
common to globular proteins. In the present study, we examined
one Ile to Ala mutant of the 3SS human lysozyme and
compared the stability change upon mutation with Ile/Leu to
Ala mutants from other proteins to obtain a general rule for
the relationship between buried water molecules and protein
stability (Shortle et al., 1990; Serrano et al., 1992; Jackson
et al., 1993; Takano et al., 1997a; Xu et al., 1998, 2001).
The results here support our previous conclusions (Takano
et al., 1997a).
The 3SS human lysozyme lacks one disulfide bond between
Cys77 and Cys95 by mutations (C77A/C95A) and is
destabilized by 20 kJ/mol, in contrast to the wild-type (4SS)
human lysozyme that has four disulfide bonds (Kuroki et al.,
1992; Takano et al., 1998). The destabilization is caused by a
substantial decrease in enthalpy, although entropy in the
denatured state could be expected to be increased owing to
the deletion of the disulfide bond. This suggests that this
protein is perturbed in both the native and denatured states. In
a previous study, a series of hydrophobic mutants from the 3SS
human lysozyme provided a general rule for the relationship
between hydrophobic effect and protein stability (Takano et al.,
1998). Therefore, the 3SS-type human lysozyme is a good
model for a generalization.
In this work, we constructed the I59A of the 3SS human
lysozyme (I59A-3SS). The residue 59 is located at a buried
site in the structure of the human lysozyme. The stability was
determined by differential scanning calorimetry (DSC) and the
structure by X-ray crystal analysis. We discuss the effect of
buried water molecules on protein stability using the results
from a 3SS-type human lysozyme and other globular proteins.
Materials and methods
Mutant proteins
Mutagenesis, expression and purification of the I59A-3SS
human lysozyme examined in this study were performed as
described previously (Takano et al., 1995). All chemicals
were of reagent grade. The protein concentration of the
mutant protein was determined spectrophotometrically using
E1%1 cm ⫽ 25.65 at 280 nm (Parry et al., 1969).
X-ray crystal analysis
The I59A-3SS human lysozyme examined was crystallized at
pH 4.5, as described previously (Takano et al., 1995; Yamagata
5
K.Takano, Y.Yamagata and K.Yutani
Table I. X-ray data collection and refinement statistics of I59A-3SS human
lysozyme
I59A-3SS
(A) Data collection
Crystal system
Space group
a, b, c (Å)
Resolution (Å)
No. of measured reflections
No. of ind. reflections
Completeness of data (%) [outer shell]
Rmergea (%) [outer shell]
I/σI [outer shell]
(B) Refinement
No. of protein atoms
No. of solvent atoms
Resolution (Å)
No. of reflections used
Completeness (%)
R-factor
Rfree
R.m.s.d. bond (Å)
R.m.s.d. angle (°)
Orthorhombic
P212121
56.83, 60.88, 32.10
1.8
50796
10772
99.0 [98.5]
4.1 [5.6]
40.1 [35.0]
1273
249
8–1.8
10565
98.9
0.179
0.256
0.009
1.49
aR
merge ⫽ 100Σ|I – ⬍I⬎|/Σ⬍I⬎.
bR-factor ⫽ Σ||F | – |F ||/Σ|F |.
o
c
o
et al., 1998). The crystal belongs to the space group P212121
with a crystal form identical with that of wild-type and 3SS
proteins (Takano et al., 1998).
I59A-3SS human lysozyme intensity datasets were
collected at 100 K at the SPring-8 on beamline 40B2 (wavelength 1.0 Å) with a Rigaku Raxis IV (Harima, Japan; Proposal
No. 2001A0090-NL-np). The data were processed with the
DENZO program (Otwinowski, 1990). The structure was
resolved by the isomorphous method and refined with the
X-PLOR program (Brunger, 1992) as described previously
(Takano et al., 1995; Yamagata et al., 1998).
Differential scanning calorimetry (DSC)
Calorimetric measurements were carried out with a DASM4
microcalorimeter. The sample buffer was 0.05 M Gly–HCl,
pH 2.35 and 2.90. The DSC data analysis was performed using
Origin software (MicroCal, Northampton, MA), as described
previously (Takano et al., 1995). The thermodynamic parameters for denaturation as a function of temperature were
calculated using the following equations (Privalov and
Khechinashvili, 1974) assuming that ∆Cp does not depend on
temperature:
∆H(T) ⫽ ∆H(Td) – ∆Cp(Td – T)
(1)
∆S(T) ⫽ ∆H(Td)/Td – ∆Cpln(Td/T)
(2)
∆G(T) ⫽ ∆H(T) – T∆S(T)
(3)
Results
Crystal structure of I59A-3SS human lysozyme
We determined the crystal structure of the I59A-3SS human
lysozyme by X-ray analysis to investigate the structural change
upon mutation. The data collection and refinement statistics
of the mutant protein are summarized in Table I. The overall
structure of the examined mutant protein was similar to that
of the 3SS protein (Takano et al., 1998). The structures of
I59A-3SS and 3SS near the mutation site are illustrated in
6
Figure 1. New water molecules were found in the mutant
structure in the space that the side-chain of residue 59 occupies
in the 3SS structure. These water molecules form a hydrogen
bond network with the protein atoms and water molecules and
the structural changes are similar to those of the I59A human
lysozyme (Takano et al., 1997a).
The coordinates of I59A-3SS were deposited in the Protein
Data Bank, accession number 1IX0.
DSC measurement of I59A-3SS human lysozyme
We examined the heat denaturation of the mutant protein by
DSC to measure the change in the conformational stability of
the I59A-3SS human lysozyme. The DSC measurement was
carried out in the acidic pH region (pH 2.35 and 2.90), where
the heat denaturation of the human lysozyme is very reversible.
Figure 2 illustrates the typical excess heat capacity curves for
I59A-3SS. Table II gives the denaturation temperatures (Td),
the calorimetric enthalpies (∆Hcal) and the van’t Hoff enthalpies
(∆HvH) of each measurement for the mutant protein. These data
and Equations 1–3 were used to calculate the thermodynamic
parameters for the denaturation of I59A-3SS and 3SS proteins
at the same temperature, 49.2°C, which is the denaturation
temperature of 3SS protein at pH 2.7 (Takano et al., 1998),
as shown in Table III. The I59A-3SS protein was destabilized
by 5.5 kJ/mol, in contrast to the 3SS protein.
Discussion
Water molecules introduced in the created cavity of I59A-3SS
It is known that the atoms in protein crystals, including
solvents, are usually well defined when they have a B-factor
in the range 0–40 Å2, but that the atoms become sufficiently
mobile or disordered that they can no longer be seen reliably
in electron density maps when the B-factor exceeds about
60 Å2 (Matthews, 1993). Three water molecules were clearly
detected in the cavity near residue 59 in both 2Fo – Fc and
Fo – Fc electron density maps of I59A-3SS. The B-factors
of these water molecules were 4.85, 15.26 and 22.17 Å2.
These results indicate that the three new water molecules in
the cavity created by the substitution were well ordered in the
I59A-3SS structure.
The new water molecules introduced in I59A-3SS formed
two or three hydrogen bonds with each other, with a protein
atom and with other buried water molecules (Figure 1). This
suggests that the buried cavity contains hydrogen-bonding
partners. Harpaz et al. reported that polar and charged atoms
occupy 38% of the buried surface in a protein interior (Harpaz
et al., 1994), indicating that there are many polar groups that
can make hydrogen bonds in the interior of proteins. This
suggests that a cavity of sufficient size to contain a water
molecule would often have ordered water molecules with
hydrogen bonds. Hubbard et al. demonstrated that most cavities
with a volume greater than 50 Å3 are hydrated (Hubbard
et al., 1994).
Stability of Ile to Ala mutants of 4SS-type and 3SS-type
human lysozymes
Table IV gives the ∆∆G values for Ile to Ala mutants of
4SS-type and 3SS-type human lysozymes. A human lysozyme
has five Ile residues and they are all completely buried
(Takano et al., 1995). The ∆∆G values of Ile to Ala mutants
of 4SS-type protein differ widely, ranging from –3.9 and
–15.5 kJ/mol (Takano et al., 1997a). However, I59A and
I106A, which are more stable than the others, have additional
Role of buried waters in protein stability
Fig. 1. Stereodrawings of the structures near the mutation site for (a) 3SS and (b) I59A-3SS human lysozymes. The broken lines and crossed circles indicate
hydrogen bonds and water molecules, respectively. The figure was generated with the ORTEP program (Johnson, 1976).
Table II. Thermodynamic parameters for denaturation of I59A-3SS human
lysozyme at different pH values
pH
Td
(°C)
∆Hcal
(kJ/mol)
∆HvH
(kJ/mol)
∆Hcal/∆HvH
2.35
2.90
36.7
47.2
233
304
243
313
0.96
0.97
Table III. Thermodynamic parameters for denaturation of the 3SS and
I59A-3SS human lysozymes at the denaturation temperature (49.2°C) of the
3SS protein at pH 2.7
Fig. 2. Typical excess heat capacity curves for an I59A-3SS human
lysozyme at pH (a) 2.35 and (b) 2.90. The increment of excess heat
capacity is 10 kJ/mol.K.
water molecules in the created cavities (Takano et al., 1997a).
This indicates that the introduced water molecules stabilize
the structure. Here, we report an Ile to Ala mutant of the
3SS-type human lysozyme. The change in stability (∆∆G)
of this mutant, I59A-3SS, relative to the 3SS protein is
–5.5 kJ/mol. This value (–5.5 kJ/mol) is comparable to that of
Td
(°C)
∆Td
(°C)
∆Cpa
(kJ/mol.K)
∆H
(kJ/mol)
∆∆H
(kJ/mol)
∆∆G
(kJ/mol)
3SSb
49.2
I59A-3SS 43.4
(0)
–5.8
7.0
6.8
342
317
(0)
–25
(0)
–5.5
a∆C
p was obtained from the slope of ∆Hcal against
bData from Takano et al. (Takano et al., 1998).
Td.
I59A (–7.2 kJ/mol) and that of I106A (–3.9 kJ/mol). I59A3SS also has new water molecules in the cavity created by
the substitution. These results demonstrate how buried water
molecules contribute to the stability of a human lysozyme.
7
K.Takano, Y.Yamagata and K.Yutani
Table IV. ∆∆G values for Ile to Ala mutants of human lysozyme
(4SS-type)
I23A
I56A
I59A
I89A
I106A
aData
∆∆Ga
(kJ/mol)
Cavity
⫺10.6
⫺15.5
⫺7.2
⫺11.3
⫺3.9
Empty
Empty
Solvated
Empty
Solvated
∆∆G
(kJ/mol)
Cavity
⫺5.5
Solvated
(3SS-type)
I59A-3SS
from Takano et al. (Takano et al., 1997a).
Table V. Average ∆∆G values for Ile/Leu to Ala mutants (buried site) of
human lysozyme (4SS-type)a and four proteins (barnaseb, T4 lysozymec,
CI2d and staphylococcal nucleasee)
Total
(A) All
Ile to Ala
23
Leu to Ala
21
Ile/Leu to Ala
44
(B) Mutants with empty cavity (structure is known)
Ile to Ala
11
Leu to Ala
7
Ile/Leu to Ala
18
(C) Mutants with solvated cavity (structure is known)
Ile to Ala
3
Ile to Ala (including I59A-3SS)
4
aData
bData
cData
dData
eData
∆∆G (kJ/mol)
⫺12.7 ⫾ 4.6
⫺14.5 ⫾ 4.4
⫺13.6 ⫾ 4.6
⫺12.6 ⫾ 2.4
⫺15.3 ⫾ 3.5
⫺13.7 ⫾ 3.1
⫺6.3 ⫾ 2.1
⫺6.1 ⫾ 1.8
from Takano et al. (Takano et al., 1997a).
from Serrano et al. (Serrano et al., 1992).
from Xu et al. (Xu et al., 1998, 2001).
from Jackson et al. (Jackson et al., 1993).
from Shortle et al. (Shortle et al., 1990).
Stability of Ile/Leu to Ala mutants of globular proteins
We summarize the average ∆∆G values for Ile/Leu to Ala
mutants of a human lysozyme (4SS-type) (Takano et al.,
1997a) and other globular proteins, barnase (Serrano et al.,
1992), T4 lysozyme (Xu et al., 1998, 2001), chymotrypsin
inhibitor 2 (CI2) (Jackson et al., 1993) and staphylococcal
nuclease (Shortle et al., 1990) in Table V. These mutation sites
are buried in the proteins. The average ∆∆G values for Ile to
Ala and Leu to Ala of all proteins were –12.7 ⫾ 4.6
and –14.5 ⫾ 4.4 kJ/mol, respectively. This verifies that both
mutations destabilize the protein structure by a decrease in
hydrophobic and van der Waals interactions (Takano and
Yutani, 2001).
Twenty-one of the 44 Ile/Leu to Ala mutant protein
structures summarized in Table V were determined. Eighteen
mutants had empty cavities and three had solvated cavities.
The average ∆∆G value of the mutants with empty cavities
was –13.7 ⫾ 3.1 kJ/mol. In contrast, the stability change was
only –6.3 ⫾ 2.1 kJ/mol for the mutants with new water
molecules. This value is similar to the ∆∆G value of I59A3SS, –5.5 kJ/mol. The average ∆∆G value of Ile to Ala
mutants with solvated cavities, including I59A-3SS, was
–6.1 ⫾ 1.8 kJ/mol. These results clearly indicate that buried
water molecules introduced by substitution partially compensate for the decrease in stability due to the decrease
in hydrophobic and van der Waals interactions. Thus, we
can conclude that buried water molecules contribute
favorably to protein stability. The difference in stability of about
7 kJ/mol between the mutants with solvated and empty cavities
8
obviously does not directly indicate the net contribution of the
water molecule, since these cavities have completely different
physical properties.
Role of buried water molecules in protein stability
We demonstrate here that Ile/Leu to Ala mutants with solvated
cavities are more stable than those with an empty cavity.
Why do buried water molecules stabilize protein structures?
Transferring an introduced water molecule from the solvent to
the interior of the protein decreases entropy. This entropic cost
is unfavorable for protein stability. Dunitz estimated the
maximum penalty to be 10 kJ/mol (Dunitz, 1994). Funahashi
et al. evaluated this effect to be about 8 kJ/mol (Funahashi
et al., 2001). In addition, there is an energy cost for the
hydration of non-polar groups in a cavity. This cost may be
minimal; it is canceled out by hydration of the ubiquitous
polar groups in the cavity (Hubbard and Argos, 1994; Buckle
et al., 1996). In contrast, buried water molecules usually have
a couple of hydrogen bonds with protein atoms and other
buried water molecules (Hubbard et al., 1994). Although it is
thought that the hydrogen bonding potential inside a protein
structure is less exploited than in the aqueous phase, many
experimental studies have demonstrated favorable contributions of hydrogen bonds to protein stability (Yamagata et al.,
1998; Funahashi et al., 1999, 2000, 2002; Takano et al., 1999a,
1999b, 2001; Pace, 2001; Pace et al., 2001; Shirley et al.,
1992; Makhatadze and Privalov, 1995). Thus, buried water
molecules stabilize a protein structure through their hydrogen
bonds. Moreover, tight packing of buried water molecules in
the interior of proteins provides better van der Waals interactions than an empty cavity (Wade et al., 1991; Takano et al.,
1997b) and thus a solvated cavity is better than an empty
cavity for protein stability. However, buried water molecules
are inferior in protein stability to non-polar protein atoms,
because even Ile to Ala mutant proteins with solvated cavities
are less stable than wild-type proteins. In contrast, a cavityfilling mutation stabilizes a protein structure (Ishikawa et al.,
1993; Akasako et al., 1997).
Conclusion
We previously reported that a water molecule in a cavity
created in the interior of a protein contributes to the stability,
based on an analysis of the change in the buried surface area
upon denaturation of the Ile to Ala/Gly mutants of a human
lysozyme (Takano et al., 1997a). We confirmed our previous
conclusion in this study using a different analysis. This analysis
demonstrates the rule of generality; buried Ile/Leu to Ala
mutations in globular proteins destabilize the proteins by about
14 kJ/mol, whereas the decrease in the stability of mutants
with solvated cavities is only about 6 kJ/mol. This indicates
that proteins partly offset the loss of hydrophobic and van der
Waals interactions by buried water molecules. Hence it is clear
that a buried water molecule contributes to the conformational
stability of a protein.
Acknowledgements
We thank Takeda Chemical Industries, Ltd (Osaka, Japan) for providing
plasmid pGEL125. This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas (C) ‘Genome Information Science’ from
the Ministry of Education, Science, Sports and Culture of Japan (Y.Y. and K.Y.).
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Received August 2, 2002; revised October 7, 2002; accepted October 7, 2002
9