Molecular Biophysics Unit, Indian Institute of Science,Bangalore 560 012, India
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Abstract |
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Keywords: protein dynamics/protein stability/temperature factors/thermophiles/X-ray structures
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Introduction |
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Various factors have been shown to contribute to the stability of proteins from thermophiles (Russell and Taylor, 1995; Querol et al., 1996
; Jaenicke and Bohm, 1998
; Ladenstein and Antranikian, 1998
). Analysis of the amino acid composition of helices in thermophilic proteins appears to indicate that Tyr, Gly and Gln are enhanced whereas Val is surpressed compared with those of mesophilic proteins (Warren and Petsko,1995
). It has also been suggested that Lys
Arg and Ser
Ala are the most frequent mutations in mesophilic to thermophilic substitutions (Arias and Argos, 1989
). The importance of electrostatic interactions (Goldman, 1995
; Hennig et al., 1995
; Yip et al., 1995
; Xiao and Honig, 1999
), increased compactness, shortening of loops, increased hydrophobicity and decreased flexibility of
-helical segments and subunit interfaces (Kelly et al., 1993
; Russell et al., 1997
) have been proposed as important factors conferring thermal stability. In the case of Che Y protein from Thermotoga maritama, thermal stability appears to be achieved by factors leading to the lowering of the entropy of unfolding (Usher et al., 1998
). Analysis of complete genome sequences has suggested loop deletion as a mechanism for thermal stability (Thompson and Eisenberg, 1999
). Surface and volume analysis has indicated that proteins from mesophilic and thermophilic origins cannot be distinguished in terms of packing criteria (Karshikoff and Ladenstein, 1998
). All these studies suggest that in thermophilic proteins stability is achieved through cooperative optimization of several subtle factors rather than any one predominant interaction.
The atomic displacement parameters (B values) determined by high-resolution X-ray crystallographic studies represent smearing of atomic electron densities around their equilibrium positions due to thermal motion and positional disorder. Analysis of B values, therefore, is likely to provide newer insights into protein dynamics, flexibility of amino acids and protein stability. Molecular dynamics studies have suggested that protein unfolding might be initiated at sites that are prone to large thermal fluctuations (Daggett and Levitt, 1992; Lazaridis et al., 1997
). Therefore, the pattern of B values determined by high-resolution X-ray crystallographic studies might contain information regarding protein stability. The correlation between experimentally observed B values and stability, unlike the contributions of various other interactions, has not been examined in any great detail.
The distribution of B values in high-resolution crystal structures has been shown to fit accurately the sum of two Gaussian functions (Parthasarathy and Murthy, 1997, 1998
). Flexibility indices of individual amino acids derived from the fitted curve reflect the dynamics of the respective amino acids. Examination of the correlation between average main-chain and side-chain B values reveals the effect of restraints used by the crystallographers for the refinement of B values and has brought out the need to have better restraints on B values (Parthasarathy and Murthy, 1999
). It has also been demonstrated that the distribution of B values reflects the special dynamic properties associated with some proteins and could possibly be used as a validation tool. In this paper, we report the analysis of B values obtained from the crystal structures of thermophilic proteins. The degrees of dispersion in the B' factors (normalized B values) associated with atoms in spheres placed at each C
atomic position in mesophilic and thermophilic proteins are comparable. Similarly, the variation of B values from the centroid towards the surface, which is likely to depend on packing density, does not appear to be significantly different in the two sets of proteins. Although the overall frequency distribution is similar, the distributions for some amino acids, especially for Ser and Thr, are different, reflecting the role played by these residues in imparting stability to thermophilic proteins. Examination of regions of high temperature factors shows that the compositions of some residues are significantly different between thermophiles and mesophiles. These observations might be related to the role played by some key residues in imparting thermal stability.
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Methods |
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Ninety-three mesophilic protein coordinates used in this analysis were chosen from the representative list (Hobohm and Sander, 1994) of PDB (Bernstein et al., 1977) entries released in November 1996. These structures have resolutions better than 2.0 Å and R factors <0.2 (Table I
). Twenty-one thermophilic structures with resolution better than 2.5 Å were used for the analysis (Table I
). Between any two of the mesophilic structures the maximum sequence similarity was 25%, while no sequence similarity criterion was applied for thermophilic structures. However, except for two structures, the thermophilic data are also non-redundant (Karshikoff and Ladenstein, 1998
).
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The B values at C atoms of each selected protein were replaced by normalized B' factors defined as, B' = (B <B>)/
(B), where <B> is the mean B value at C
atoms and
(B) is their standard deviation. Frequencies of residues in 0.5 unit ranges in B' factors were counted. Various frequency distributions for individual amino acids and individual proteins and the overall distribution were counted and fitted analytically by least-squares minimization to the sum of two Gaussian functions (Parthasarathy and Murthy, 1997
, 1998
), f = k1exp[k2(B'B1)2 + k3exp[k4(B'B2)2], where k1, k2, k3, k4, B1 and B2 are parameters defining the two Gaussians. The constants for the second Gaussian were determined after fitting the first Gaussian to stabilize the minimization. The areas under the two Gaussians, A1 and A2, are given by A1 = k1(
/k2)
and A2 = k3(
/k4)
. The fractional areas under the second Gaussian, p = A2/(A1 + A2), for individual amino acids, representing the propensity to occur with high B value, were calculated.
Distribution of high B values
Amino acids with B values greater than <B> + 0.5(B) were considered as high B value residues. The amino acid compositions of residues with high B values in mesophilic and thermophilic proteins were determined and compared. The numbers of stretches of consecutive high B value residues of length 15 were counted. A similar analysis was also performed with a high B value threshold of <B> + 0.75
(B).
Dispersion of B values in spheres placed at C positions
Spheres of radius 5.0 and 7.5 Å were placed at each C atom. The relatedness of the B values of atoms in these spheres was analysed by calculating the r.m.s. deviation of their B' factors. The frequency distributions of these r.m.s. values were determined. Also, a plausible correlation that might exist between the mean B' factors in these spheres and the corresponding atomic packing was examined.
Variation of B values with distance from centroid
For each protein, the radius of gyration was calculated as Rg = (|ri rc|2/n)
, where ri and rc represent the positions of C
atoms and the centroid of the molecule, respectively. Mean B' factors in spherical shells of radius expressed in terms of Rg were computed for thermophilic and mesophilic proteins.
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Results |
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Figure 1 shows the overall frequency distribution of B' factors for mesophilic and thermophilic proteins. The plots represent curves fitted as the sum of two Gaussian functions and correspond to 30 960 amino acids for mesophiles and 10 469 amino acids for thermophiles. The six parameters characterizing the double Gaussian function are very similar for the two curves. The fractional areas under the second Gaussian, the p-values, are 0.357 and 0.361 for mesophilic and thermophilic proteins, respectively. Table II
gives the p-values for individual amino acids. It can be seen that Glu, Leu, Tyr and Gln have higher p-values in thermophiles than mesophiles whereas Cys, Asn, Pro, Arg and Ser have lower p-values.
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The frequency of occurrence of consecutive high B values [defined as <B> + 0.5(B)], stretches of length 15, were determined for mesophilic and thermophilic proteins. Table III
lists the relevant statistics. It can be seen that there is no substantial difference in terms of the length distribution or frequency of amino acids found in these stretches between thermophiles and mesophiles. This observation suggests that the thermophilic and mesophilic proteins do not differ in the occurrence of segments of high or low mobilities.
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Figure 2a shows the scatter plot of overall amino acid composition in mesophiles and thermophiles. Table IV
gives the corresponding statistics. Figure 2b and c
show scatter plots of high B value residues. As can be seen in Figure 2a
and Table IV
, the overall amino acid compositions are very similar in mesophiles and thermophiles (correlation coefficient 0.89). The average correlation coefficient, however, between compositions of high B value residues in mesophiles and thermophiles is 0.77. The residues Glu, Lys, Ser and Thr are outliers in these plots (Figure 2b and c
and Table IV
). Notably, the percentage Glu residues in high B value regions is nearly twice and that of Lys is nearly 1.5 times higher in thermophiles than mesophiles. In contrast, the percentages of Ser and Thr in high B value regions of thermophiles are decreased by half (Table IV
). These are also related to larger p-values (Table II
) for Glu, Lys and smaller p-values for Ser and Thr in thermophiles. Similar observations were made when the B values of whole residues instead of those associated with C
alone were examined (data not shown).
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It is expected that in the well packed interior of proteins, atoms in close proximity will have correlated displacements, which will be reflected in the B values. If thermophiles have better packed interiors than mesophiles, atoms in spherical volumes around a large number of C positions will have low r.m.s. B values in thermophiles than mesophiles. Figure 3a and b
show the frequency distribution of r.m.s. B' factors in these spherical regions for mesophiles and thermophiles, for radii of 5.0 and 7.5 Å, respectively. There is no significant difference in peak position or frequency in each bin between mesophilic and thermophilic proteins. This agrees with an earlier report that mesophilic and thermophilic proteins do not differ significantly with respect to packing interactions (Karshikoff and Ladenstein, 1998
).
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It is known that B values tend to increase continuously from the core of the protein to its surface (Bhaskaran and Ponnuswamy, 1988). It is of interest, therefore, to compare the increment in B values from the core of the protein to the exterior between mesophiles and thermophiles. Hence the increment in B' factors in spherical shells of radius corresponding to a specified fraction of the radius of gyration of the molecule was examined. If the interiors of thermophiles are better packed than those of mesophiles, the slope of this incremental curve will be smaller for thermophiles (Figure 4
). It is clear from the plot that the two curves are almost identical except for fluctuations observed in shells near the outer surface of mesophiles. This fluctuation at outer shells shown by the mesophiles may be due to loops extending out from the rest of the protein.
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Discussion |
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Better hydrogen bonding networks, electrostatic interactions and better internal packing have been suggested by various investigators as factors contributing to the stability of thermophilic proteins (Russell and Taylor, 1995; Querol et al., 1996
; Jaenicke and Bohm, 1998
; Ladenstein and Antranikian, 1998
). Karshikoff and Ladenstein (1998) have analysed the packing density in mesophilic and thermophilic proteins and concluded that packing density is not a dominant factor contributing to the thermal stability. However, contradictory results were obtained when glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase from mesophilic and thermophilic organisms were compared for their accessible surface area (Korndorfer et al., 1997
; Knapp et al., 1997
). Analyses of variations in B' factors around spheres at C
atoms and increment of B' factors from the core to the surface of proteins, presented here, show that thermophlic proteins are not very different from mesophlic proteins in these aspects. Packing differences, if any, are thus not reflected in the atomic displacement parameters.
The most significant observation in the present analysis is that Glu and Lys are enhanced whereas Ser and Thr are suppressed in high B value regions of thermophiles in comparison with mesophiles. The juxtaposition of these four residues is perhaps important in imparting thermal stability. These residues may be suitable candidates for site-specific mutations leading to enhanced stability. This also suggests that mutation of high B value Ser and Thr could lead to an improvement of thermal stability. The mutational experiments on lactate dehydrogenase by Kotik and Zuber (1993) and streptococcal protein G ß1 domain (Malakauskas and Mayo, 1998) involving Ser and Thr residues have led to considerable increases in thermal stability, suggesting that the conclusions drawn from the present analysis are likely to be significant. These results also suggest that the `traffic rules' of amino acid replacements need to be revised with reference to amino acid flexibility.
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Acknowledgments |
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Notes |
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References |
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Bernstein,F.C., Koetzle,T.F., Williams,G.J.B., Meyer,E.F.,Jr, Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchui,T. and Tasumi,M. (1997) J. Mol. Biol., 112, 535542.
Bhaskaran,R. and Ponnuswamy,P.K. (1988) Int. J. Pept. Protein Res., 32, 241255.[ISI]
Bohm,G. and Jaenicke,R. (1994) Int. J. Pept. Protein Res., 43, 97106.[ISI][Medline]
Daggett,V. and Levitt,M. (1992) Proc. Natl Acad. Sci. USA, 89, 51425146.[Abstract]
Goldman,A. (1995) Structure, 3, 12771279.[ISI][Medline]
Hennig,M., Darimont,B., Sterner,R., Kirschner,K. and Jansonius,J.N. (1995) Structure, 3, 12951306.[ISI][Medline]
Hobohm,U. and Sander,C. (1994) Protein Sci., 3, 522524.
Jaenicke,R. and Bohm,G. (1998) Curr. Opin. Struct. Biol., 8, 738748.[ISI][Medline]
Karshikoff,A. and Ladenstein,R. (1998) Protein Engng, 11, 867872.[Abstract]
Knapp,S., de Vos,W.M., Rice,D. and Ladenstein,R. (1997) J. Mol. Biol., 267, 916932.[ISI][Medline]
Kelly,C.A., Nishiyama,M., Ohnishi,Y., Beppu,T. and Birkoft,J.J. (1993) Biochemistry, 32, 39133922.[ISI][Medline]
Korndorfer,I., Steipe,B., Huber,R., Tomschy,A. and Jaenicke,R. (1997) J. Mol. Biol., 246, 511521.
Kotik,M. and Zuber,H. (1993) Eur. J. Biochem., 211, 267280.[Abstract]
Ladenstein,R. and Antranikian,G. (1998) Adv. Biochem. Engng Biotechnol., 61, 3785.
Lazaridis,T., Lee,I. and Karplus,M. (1997) Protein Sci., 6, 25892605.
Malakauskas,S.M. and Mayo,S.L. (1998) Nature Struct. Biol., 5, 470475.[ISI][Medline]
Matthews,B.W. (1995) Adv. Protein Chem., 46, 249278.[ISI][Medline]
Parthasarathy,S. and Murthy,M.R.N. (1997) Protein Sci., 6, 25612567.
Parthasarathy,S. and Murthy,M.R.N. (1998) Protein Sci., 7, 525.
Parthasarathy,S. and Murthy,M.R.N. (1999) Acta Crystallogr., D55, 173180.[ISI]
Querol,E., Perez-Pons,J.A. and Villarias,A.M. (1996) Protein Engng, 9, 265271.[Abstract]
Russell,R.J.M. and Taylor,G.L. (1995) Curr. Opin. Biotechnol., 6, 370374.[ISI][Medline]
Russell,R.J.M., Ferguson,J.M.C., Hough,D.W., Danson,M.J. and Taylor,G.L. (1997) Biochemistry, 36, 99839994.[ISI][Medline]
Thompson,M.J. and Eisenberg,D. (1999) J. Mol. Biol., 290, 595604.[ISI][Medline]
Usher,K., De la Cruz,A., Dahlquist,F., Swanson,R., Simon,M. and Remington,S. (1998) Protein Sci., 7, 403412.
Warren,G.L. and Petsko,G.A. (1995) Protein Engng, 9, 905913.[Abstract]
Xiao,L. and Honig,B. (1999) J. Mol. Biol., 289, 14351444.[ISI][Medline]
Yip,K.S.P., Stillman,T.J., Britton,K.L., Artymuick,P.J., Baker,P.J., Sedelniova, S.E., Engel,P.C., Pasquo,A., Chiaralauce,R., Consavi,V., Scandurra,R. and Rice,D.W. (1995) Structure, 3, 11471158.[ISI][Medline]
Received April 23, 1999; revised September 12, 1999; accepted October 1, 1999.