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Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding. The enzyme DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability. The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding. The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C. The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C. There is no flattening of the stability profile. Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability. TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates. The unfolding reaction is much slower (ca. 10(8) times) compared to DHFR from Escherichia coli (EcDHFR). In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable. Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude. Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer.

Structural analysis of TmLDH and comparison of the enzyme to moderately thermophilic and mesophilic homologs reveals a strong conservation of both the three-dimensional fold and the catalytic mechanism. Going from lower to higher physiological temperatures a variety of structural differences can be observed: an increased number of intrasubunit ion pairs; a decrease of the ratio of hydrophobic to charged surface area, mainly caused by an increased number of arginine and glutamate sidechains on the protein surface; an increased secondary structure content including an additional unique 'thermohelix' (alphaT) in TmLDH; more tightly bound intersubunit contacts mainly based on hydrophobic interactions; and a decrease in both the number and the total volume of internal cavities. Similar strategies for thermal adaptation can be observed in other enzymes from T. maritima.

Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel-permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02 -50 mg/ml) and temperature (20-SOOC). Rechromatography confirms this finding : the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species ; increased temperature favors octamer formation.Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation cause irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion : image processing shows that the dumb-bell-shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.Keywords: hyperthermophiles ; lactate dehydrogenase; quaternary structure ; stability ; Thermotoga muritimu.Lactate dehydrogenases (LDH) are dimeric or tetrameric enzymes that require their native quaternary structure for catalysis (Jaenicke, 1974). As has been shown by denaturation-renaturation experiments, using domain fragments and intact subunits of porcine skeletal muscle LDH, tertiary and quaternary interactions provide increments of stability which in toto yield the exceedingly high value for the free energy of stabilization observed for the enzyme (Miiller et al., 1982;Pfeil, 1986; Opitz et al., 1987;Jaenicke, 1991). This supports the general view that protein stability is accomplished by the cumulative effect of covalent and non-covalent bonds at the various levels of the hierarchy of protein structure (Vita et al.. 1989;Jaenicke, 1996).There are various enzymes from thermophilic (and hyperthermophilic) organisms that exhibit anomalously high states of association, which suggests thermal adaptation of proteins to be attributable to higher states of subunit assembly. For example, pyruvate dehydrogenase from Bacillus stearothermophilus (To,, ~6 5°C ) contains four times as many polypeptide chains comCorrespondence ta R. Jaenicke, lnstitut