1Department of Chemistry and 2Department of Biology, California State Polytechnic University at Pomona, Pomona, CA 91768, USA
3 To whom correspondence should be addressed. e-mail: drlivesay{at}csupomona.edu
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Abstract |
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Keywords: electrostatics/salt bridges/structural bioinformatics/thermostability
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Introduction |
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Several recent efforts have attempted to identify the most efficient method of conferring enhanced thermostability to a mesophilic protein structure. Early efforts in this direction have concentrated on engineering new structural tethers (covalent and non-covalent interactions) within the protein structure (Perry et al., 1989; Scholtz and Baldwin, 1992
; Serrano et al., 1992
; Bryson et al., 1995
; Matthews, 1995
; Pace, 1995
; Cordes et al., 1996
; Lehmann et al., 2000
; Lehmann and Wyss, 2001
). This approach is well justified because it is becoming increasingly clear that protein folding is a hierarchical process and thus is mostly driven by local interactions (Baldwin and Rose, 1999a
,b). However, recent results reveal that the complex electrostatic properties on the proteins surface can also contribute significantly to its thermostability. Several recent studies have successfully increased mesophilic protein stability through mutagenesis of a single solvent exposed residue, presumably through optimization of its electrostatic surface (Grimsley et al., 1999
; Loladze et al., 1999
; Perl et al., 2000
; Spector et al., 2000
; Strop and Mayo, 2000
; Martin et al., 2001
; Pedone et al., 2001
; Perl and Schmid, 2001
; Loladze and Makhatadze, 2002
). Additionally, our comprehensive in silico screening of surface mutants confirms that optimization of the electrostatic surface is a robust strategy for conferring thermostability to mesophilic proteins (Torrez et al., 2003
). From these results, it is apparent that surface electrostatics are intimately related to protein stability, and that optimization of the electrostatic surface is an attractive mechanism for conferring increased thermostability to a protein structure. Thus, we now inquire to the extent that nature has also employed similar design mechanisms.
There is a growing list of anecdotal evidence suggesting that optimization of the electrostatic surface of thermophilic proteins is a robust evolutionary theme (Fukuchi and Nishikawa, 2001). Comparisons of the amino acid composition of thermophilic versus mesophilic proteomes reveal significantly more charged residues (especially Arg, Lys and Glu) within thermophilic proteins (Chakravarty and Varadarajan, 2002
; La et al., 2003
). The charge composition differences are generally ascribed to greater numbers of stabilizing ion pairs in thermophilic structures, yet it is unclear what percentage of these differences are specific chargecharge interactions within the core (salt bridges) versus more promiscuous long-range interactions on the protein surface. Here, we report a clear preference for stabilizing solvent exposed (opposite charge) ion pairs on the surface thermophilic protein structures.
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Materials and methods |
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Our program, Surface Finder, iteratively evaluates the solvent accessibility of each atom (excluding hydrogens). Here, if an acid or base charged atom is deemed solvent accessible, then that residue is included in the ion pair distance analysis. Charged atoms are defined as atoms explicitly carrying the formal charge (NZ of Lys) or atoms central to resonance-equivalent charged atoms (CG of Asp, CD of Glu, CZ of Arg and CD2 of His). All distances reported are linear between charged atom pairs; no attempt to correct for curvature of the proteins surface has been made, although future work will. Surface Finder is provided freely to the academic community upon request.
Electrostatic potential maps
Electrostatic potential maps are calculated using the PoissonBoltzmann equation solver within MOE.2. The protein is centered on a 33x33x33 cubic grid. A solvent dielectric constant of 80 and a protein dielectric constant of 4 are used in all electrostatic potential map calculations, which are standard values in electrostatic potential map calculations (Sharp and Honig, 1990). Protein partial charges are taken from the CHARMM parameter set (Brooks et al., 1983
). The temperature is 300 K, the counter ion radius is 1.4 Å, the ionic strength is 0.15 M, and the protein concentration is 0.001 M. Electrostatic potentials are rendered in blue and red at ±1.5 kcal/mol/e, respectively.
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Results and discussion |
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The maximum number of acidbase pairs for both mesophilic and thermophilic structures is centered at 4.5 Å, which is routinely considered the upper limit for salt bridges. On the other hand, the number of acidacid and basebase pairs increases roughly linearly with distance. Cursory examination of the structures reveals that acidacid and basebase pairs at larger distances are often shielded by an acidbaseacid or baseacidbase triplet, and thus not very destabilizing.
These points are exemplified by comparisons of the 2-dehydro-3-phosphooctanate from Escherichia coli and the thermophilic bacterium Aquifex aeolicus (Asojo et al., 2001; Wang et al., 2001
). The enzyme is a member of the ubiquitous TIM-barrel fold family, with the active site at the top of the structure (Nagano et al., 2002
). Despite vastly different optimal growth temperatures of the two organisms (37 versus 80°C), traditional comparisons of the two orthologous structures reveal no obvious stabilizing mechanism. The structures possess nearly identical secondary and tertiary structures, and similar numbers of intramolecular contacts. The thermophilic ortholog has 46 (non-secondary structure) hydrogen bonds and 16 salt bridges, whereas the mesophilic ortholog has 41 and 18, respectively. Only Ramachandran analysis provides any appreciable differences between the two structures stability (Figure 2B).
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Conclusions
The results reported here confirm that optimization of the electrostatic surface is a robust evolutionary mechanism for increasing thermostability, which had been previously suggested based on anecdotal evidence. Comparison of 127 orthologous mesophilicthermophilic protein groups clearly indicates a preference for stabilizing acidbase pairs on the surface of thermophilic proteins. Compared with positions buried in the core, stabilizing surface mutations are less likely to disrupt the tertiary structure, and thus more likely to be evolutionarily selected. Therefore, we believe that these results, in addition to being theoretically interesting, will facilitate identification of charge-altering mutations likely to increase the stability of particular protein structures.
Supplementary data
A complete list of PDB i.d.s, their thermal classification, and raw data indicating the number of acidacid, acidbase and basebase ion pairs identified are available as supplementary data at Protein Engineering online.
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Acknowledgements |
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References |
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Received July 23, 2003; revised October 14, 2003; accepted October 21, 2003