1 Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 and 3 Graduate School of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi, Kumamoto 862-0973, Japan 4 Present address: Graduate School of Sciences, Kwansei Gakuin University, Gakuen, Sanda, Hyogo 669-1337, Japan
To whom correspondence should be addressed. Present address: Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ktakano{at}mls.eng.osaka-u.ac.jp
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Abstract |
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Keywords: cavity/human lysozyme/mutant protein/protein stability/water molecule
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Introduction |
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Many theoretical studies have been conducted to understand 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.
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Materials and methods |
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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 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 GlyHCl, 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:
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Results |
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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 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
).
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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 vant 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.
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Discussion |
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It is known that the atoms in protein crystals, including solvents, are usually well defined when they have a B-factor in the range 040 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 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 I59A (-7.2 kJ/mol) and that of I106A (-3.9 kJ/mol). I59A-3SS 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.
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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
).
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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 cavity-filling 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.
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Acknowledgments |
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Received August 2, 2002; revised October 7, 2002; accepted October 7, 2002.