Protein Engineering, Vol. 14, No. 8, 525-528,
August 2001
© 2001 Oxford University Press
COMMUNICATION |
A new scale for side-chain contribution to protein stability based on the empirical stability analysis of mutant proteins
1 Institute for Protein Research, Osaka University, Yamadaoka, Suita,Osaka 565-0871, Japan
E-mail: yutani{at}protein.osaka-u.ac.jp
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Abstract |
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 Top Abstract IntroductionCalculation of {Delta}{Delta}G...Side-chain contribution to...Side-chain contribution of...Side-chain contribution of...References |
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The hydrophobicity scales for amino acid side chains based on the transfer Gibbs energy (
Gtrans) of amino acids from non-aqueous phases to water have been widely used to estimate the contribution of buried side chains to the conformational stability of proteins. In this paper, we propose a new scale for the side-chain contribution to protein stability, which is derived from data on protein denaturation experiments using systematic and comprehensive mutant proteins. In the experiments, the contribution of some physical properties were quantitatively determined as parameters in a unique equation representing the stability change (G) of mutant proteins as a function of the structural changes due to the mutations. These parameters are able conveniently to provide a scale for the side-chain contribution to protein stability. This new scale also has the advantage over the previously reported hydrophobicity scales of residues with the contributions of hydrogen bonds or secondary structural propensity. It may find practical application in algorithms for the prediction of protein structures.
Keywords: hydrophobicity/mutant protein/protein denaturation/protein stability/stability scale/transfer Gibbs energy
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Introduction |
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TopAbstractIntroductionCalculation of {Delta}{Delta}G...Side-chain contribution to...Side-chain contribution of...Side-chain contribution of...References |
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The hydrophobic effect is believed to play a major role in organizing the self-assembly of globular proteins (Kauzmann, 1959
; Dill, 1990). To quantify this effect, many hydrophobicity scales for the amino acid residues have been proposed (Nozaki and Tanford, 1971; Wolfenden et al., 1981; Fauchere and Pliska, 1983; Rose et al., 1985; Damodaran and Song, 1986; Kawashima et al., 1999). These scales are based on the transfer Gibbs energy (Gtrans) of the amino acid residues from a non-aqueous phase to an aqueous phase. The octanol scale taken from Fauchere and Pliska (1983) is probably the most commonly used for estimating the contribution of buried non-polar side chains to the conformational stability of proteins.
A comparison of several scales, however, reveals that significant differences exist among them. Especially the values for polar or aromatic side chains are highly dependent on the non-aqueous phase (Karplus, 1997). There is then the question of which non-aqueous phase best characterizes the protein interior (Dill, 1990) and even some doubt as to whether they can be applied to protein denaturation (Vajda et al., 1995). This evidence suggests that we need a hydrophobicity scale of amino acid residues obtained from data on protein denaturation experiments. To obtain hydrophobicity parameters from protein experiments, we will introduce an alternative method based on the quantifiable changes in protein stability due to mutations (Sharp et al., 1991; Vajda et al., 1995).
Recently, it has been proposed that the stability change (G) of each mutant human lysozyme can be precisely represented in a unique equation by considering the conformational changes due to the mutations (Funahashi et al., 1999; Takano et al., 1999b). This G calculation provides each contribution of several physical properties, such as the hydrophobic effect and hydrogen bonds, to protein stability and it is compatible with other proteins (Funahashi et al., 2001).
In this paper, we propose a new scale for the side-chain contribution to protein stability, which is derived from the empirical G calculation on mutant proteins (Funahashi et al., 2001). This new scale presented here is more valuable than the previous hydrophobicity scales; it is based on more precise data from experimental studies on proteins and it is also able to show the contribution of residues with hydrogen bonds and residues in secondary structures.
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Calculation of G of mutant proteins
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TopAbstractIntroductionCalculation of {Delta}{Delta}G...Side-chain contribution to...Side-chain contribution of...Side-chain contribution of...References |
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It has been proposed that the changes in stability (
G, kJ/mol) of each mutant protein are represented by a unique equation, as expressed by Equation 1 (Funahashi et al., 2001). In this equation, the contributions of the hydrophobic effect (GHP), side-chain conformational entropy (Gconf), hydrogen bonds (GHB), water molecules (GH2O), secondary structural propensity (Gpro: Gpro
and Gproß) and cavity volume (Gcav) to the stability are represented by each parameter in terms of the conformational change due to the mutation, as expressed by Equations 2–7. These parameters can be generally applied to globular proteins (Funahashi et al., 2001).
 | (1) |
 | (2) |
 | (3) |
 | (4) |
 | (5) |
 | (6) |
 | (7) |
ASANP and ASAP represent the differences in the ASA (accessible surface area, Å2) of the non-polar (C and S) and polar (N and O) atoms, respectively, of all residues upon denaturation; Sconf is the difference in the side-chain conformational entropy upon denaturation defined by Doig and Sternberg (1995); rpp, rpw and rww are the length (Å) of the intramolecular, protein–water and wate–water hydrogen bonds, respectively, introduced/deleted due to mutation; NH2O is the number of water molecules introduced; P and Pß are the -helix and ß-sheet propensities, respectively, of the residue defined by Chou and Fasman, (1978) [revised by Koehl and Levitt (1999)]; and Vcav represents the cavity volume (Å3). The coefficients in Equations 2
and 4–7 have been estimated by a least-squares fit of each G (Equation 1) to the experimental G values using the stability–structure database of mutant proteins (Funahashi et al., 2001). The meaning of each parameter has been discussed in the previous papers by Funahashi et al. (1999, 2001). |
Side-chain contribution to protein stability (Gaa) based on G calculation
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The parameters of various stabilization factors used in the
G calculation described above are appropriate for a basic understanding of protein stability. We are able to extract the general contributions of each amino acid side chain for the conformational stability of globular proteins relative to Gly (Gaa) from the parameters of the hydrophobic effect and side chain entropy:
 | (8) |
Gaa value is calculated using the ASA value of the amino acid residue (Miller et al., 1987) and the entropic effect of the amino acid residue (Doig and Sternberg, 1995), as shown in Table I. As the Gaa values include the entropic effect, the Gaa values might not correspond to the Gtrans values (hydrophobicity), so they represent the contributions of each amino acid side chain to the conformational stability of the globular proteins. The GHP values might correspond to the Gtrans values. The Gtrans values of a vapor (Wolfenden et al., 1981), cyclohexane (Radzicka and Wolfenden, 1988) and octanol (Fauchere and Pliska, 1983) to water are also listed in Table I. The Gtrans value of a vapor to water is different from those of the other scales, because there is little van der Waals interaction in the vapor phase.
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In the
Gaa values, all residues (except for Gly) compensate the entropic loss of the side chain with the hydrophobic effect, resulting in a more positive contribution to the stability than the Gly residue. This means that even polar side chains contribute favorably to the protein stability through the hydrophobic effect from their non-polar atoms. This point is different from previous transfer values; usually polar residues would contribute negatively to the stability. |
Side-chain contribution of residues with hydrogen bonds to protein stability (GaaHB)
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The side chains of buried polar residues usually form hydrogen bonds with the protein atoms or water molecules. The hydrogen bonds contribute to the protein stability (Myers and Pace, 1996
; Yamagata et al., 1998; Takano et al., 1999a,b). We are also able to estimate the contribution of residues with hydrogen bonds (GaaHB) using the parameters of the hydrogen bonds:
 | (9) |
GaaHB values of each amino acid residue with several hydrogen bonds are summarized in Table II.
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In Table II
, the polar residues with hydrogen bonds contribute to the stability of proteins, similarly to hydrophobic residues. This means that a protein structure is stabilized not only by buried hydrophobic residues but also by buried polar residues with hydrogen bonds. For example, for the Val and Thr residues, which are isosteric residues, the Thr residue with one intramolecular hydrogen bond has a contribution (14.3 kJ/mol) comparable to the Val residue (15.3 kJ/mol). Furthermore, when a Thr residue has two intramolecular hydrogen bonds, it contributes (21.7 kJ/mol) more than a Val residue. In fact, a Thr residue forms about two hydrogen bonds on average in protein structures (McDonald and Thornton, 1994). Recently, Pace (Pace, 2001) has pointed out in another way that burial of a polar residue contributes more to protein stability than burial of a non-polar residue. |
Side-chain contribution of residues on secondary structures to protein stability (Gaa and Gaaß)
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TopAbstractIntroductionCalculation of {Delta}{Delta}G...Side-chain contribution to...Side-chain contribution of...Side-chain contribution of...References |
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In secondary structures, the
-helix and ß-sheet, certain amino acids are found more frequently, but others are found less frequently (Chou and Fasman, 1978). This tendency is known as -helix or ß-sheet propensity. These propensities affect the protein stability (Pace and Scholtz, 1998; Street and Mayo, 1999), suggesting that the stability scales of residues in the secondary structures are different to those in the other regions. We also calculated the contributions of residues in the -helix (Gaa) and in the ß-sheet (Gaaß) relative to Gly using the parameters of the secondary structural propensities:
 | (10) |
 | (11) |
P and Pß are the -helix and ß-sheet propensities, respectively, of the residue relative to Gly defined by Chou and Fasman (Chou and Fasman, 1978) [revised by Koehl and Levitt (1999)]. The Gaa and Gaaß values are summarized in Table III. If a residue on a secondary structure forms hydrogen bonds, the contribution of hydrogen bonds could definitely be added as described above.
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Conclusion
It is not easy to predict the tertiary structure of a protein from the amino acid sequence, although it is one of the most important subjects in life science research. One problem is that the computational approaches to predicting protein structures mostly use the hydrophobicity scales based on the transfer Gibbs energy as the contribution of the amino acid residues to protein stability. We now propose a new scale for the side chain contribution to stability based on an experimental study of proteins. This scale is different to the previous scales, especially for polar residues. We expect that this scale would be useful to theoretical researchers in improving structural predictions. Furthermore, the contributions of residues in various environments, such as residues with hydrogen bonds or residues in secondary structures, are also estimated. This scale will also contribute significantly to improving the protein stability.
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Notes |
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1 To whom correspondence should be addressed. E-mail: yutani{at}protein.osaka-u.ac.jp
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Acknowledgments |
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This work was supported in part by Fellowships from the Japan Society for the Promotion of Science for Young Scientists (K.T.) and 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 (K.Y.).
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Received February 9, 2001; revised May 25, 2001; accepted July 8, 2001.
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