The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild-type phosphoglycerate kinase PGK-TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D-structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermo-philes. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability , were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two ther-mophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process. Proteins 1999;37:441-453. 1999 Wiley-Liss, Inc.

We undertook a first detailed comparative analysis of the refolding kinetics of ribonuclease A (RNase A) by time-resolved Fourier transform infrared spectroscopy. The refolding process was initiated either by applying a temperature jump on the thermally denatured protein or by rapid dilution of a concentrated [13C]urea solution containing the chemically unfolded protein. The dead time of the injecting and mixing devices and the time-resolution of the spectrometer permitted us to monitor the refolding kinetics in a time range of 100 ms to minutes. The infrared amide I' band at 1631 cm-1 was used to directly probe the formation of beta-sheet structure during the refolding process. The aromatic ring stretching vibration of tyrosine at 1515 cm-1 was employed as a local monitor that detects changes in the tertiary structure along the folding pathway. The comparative analysis of the kinetics of the beta-sheet formation of chemically and thermally denatured ribonuclease A revealed similar folding rates and amplitudes when followed under identical refolding conditions. Therefore, our kinetic infrared studies provide evidence for a high structural similarity of urea-denatured and heat-denatured RNase A, corroborating the conclusions derived from the direct comparison of the infrared spectra of thermally and chemically denatured RNase A under equilibrium conditions [Fabian, H., & Mantsch, H.H. (1995) Biochemistry 34, 13651-13655]. In detail, the kinetic infrared data demonstrate that in the time window of 0.1-30 s approximately 40% of the native beta-sheet structure in RNase A is formed in the presence of 0.6 M urea at pH* 3.6, indicating that up to 60% of the beta-structure is formed out of the time window used in this study. Temperature jump experiments in the absence of chemical denaturants exhibited faster and more complex refolding kinetics. In addition, differences in the time constants of refolding derived from the amide I' band at 1631 cm-1 and from the tyrosine vibration at 1515 cm-1 were observed, indicating that the formation of secondary structure precedes the formation of stable tertiary contacts during the refolding of RNase A.

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.

The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA⅐ CcdB⅐DNA complex with a CcdA:CcdB ratio of one. This (CcdA 2 CcdB 2 ) n complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.