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The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, a group from UC Berkeley (Biochemistry 38(1999):3831-6 ) has recently determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.
Looking through some of the recent literature, it appears that the increased thermodynamic stability seems to be the currently accepted explanation and several groups claim that extremozymes are approximately 5 kcal/mol more stable than their mesophilic counterpart as judged from equilibrium denaturation studies. It seems that a higher number or more favourable salt bridges make a major contribution to thermostability. There are, however, alternative explanations brought forward by other groups, e.g. stressing the importance of disulphide bonds, although many of the extremozymes do not contain any. It can be expected that this will remain a widely debated question for several years.
I also recommend a recent review by R. Ladenstein (Karolinska Universitet, Stockholm) for an excellent overview of the state-of-the-art in extremozyme enzymology (Adv. Biochem. Eng. Biotechnol. 61(1998)37-85) .
It is obvious, that there have to be some stabilizing interactions that yield a higher Tm for protein-DNA interactions in thermophiles than in mesophiles. Recently, a Japanese group has identified the interaction sites between DNA and the DNA-binding protein HU from the thermophile Bacillus stearothermophilus (BstHU). Replacing several of the residues interacting with DNA by the residues occuring at the same positions in the homologue from a mesophile B. subtilis (BsuHU), they could reduce the thermostability of the interaction. On the other hand, they could introduce thermostabilizing mutations into BsuHU and create a mutant that had a similar Tm as BstHU (J.Biol. Chem. 273(1998):19982-7).
In another study on histone-DNA interactions in a member of the Thermococcus family, footprinting studies suggest a different organization of the protein along the DNA compared to mesophilic relatives.
Obviously, a greater G/C content and thus a higher Tm for double stranded DNA is encountered in genes from thermophiles. This can, by the way, sometimes cause problems when amplifying those genes by PCR. In addition, it has recently been suggested that there is also an important contribution of salt in protecting DNA from denaturation at high temperatures, although most studies simply state the G/C content as reason for thermostability.
Another of the open questions. As thermodynamic equilibria are quite dependend on temperature, it is conceivable that interactions in the regulation of metabolism might be very different in thermozymes. A number of studies on the enzyme kinetics of enzymes from various thermophiles and hyperthermophiles suggest similar kinetic parameters at optimal reaction conditions as in the mesophilic counterparts of those enzymes. So far, very little has been done in studies of allosteric regulation in thermophiles (there are some studies at high pressure for enzymes from deep-sea organisms, though). One of the few exceptions is a recent study on pyruvate kinase from Bacillus stearothermophilus that exhibits cooperativity of substrate binding, although the simple two-state model that describes this process in mesophiles does not apply in this case and a more complex model of interaction must exist (J Mol Biol 276(1998):839-51).
In addition to the references to papers above, I recommend an article that appeared in Scientific American in 1997 and is available via the web at http://www.sciam.com/0497issue/0497marrs.html and the very recent review on Extremozymes by Hough that appeared in Curr. Opin. Chem. Biol. 3(1999):39-46.
Back to The Macromolecular Crystallography Home Page
LLNL Disclaimer
This World Wide Web site conceived and maintained by
Bernhard Rupp (br@llnl.gov)
Last revised November 10, 1999 12:10
UCRL-MI-125269