Proteins and particularly enzymes are generally believed to be vulnerable structures sensitive to environmental changes, however, there are some exceptions. Extreme thermophilic microorganisms have an optimum growth temperature above 70 °C, while psychrophilic organisms are capable of living below 0 °C. Most enzymes isolated from thermophilic and psychrophilic sources are highly homologous in structure and function to those of mesophilic origin. The same building blocks, the same underlying physical principles and highly similar folds are used in these extremophilic enzymes. This observation raises the question: if heat resistant and cold tolerant proteins can also be constructed for a given function, using the same building elements and principles, why most enzymes work optimally at the edge of their stabilities.

We observed that the activity profile of enzymes does not follow the Arrhenius equation: the activation energy of enzyme catalyzed reactions decreases at increased temperatures. The answer for these observations might lie in the dynamic nature of the molecular events associated with enzymatic catalysis. Conformational flexibility kept at optimum level is required for full enzymatic activity. The loss of activity of thermophilic enzymes at low temperatures, where most related enzymes are fully active might be the consequence their increased conformational rigidity required to stabilize the protein against heat denaturation.

To check this hypothesis we perform comparative activity, thermostability and conformational flexibility studies using a thermophilic-mesophilic-psychrotrophic enzyme triplet. The model enzyme was 3-isopropylmalate dehydrogenase (IPMDH) isolated from Thermus thermophilus, Escherichia coli and Vibrio sp. I5 (Figure 1). These enzymes were cloned and expressed and purified to homogeneity using E. coli expression systems.

The reaction catalyzed by IPMDH was monitored by UV spectrophotometry at different temperatures from 14ºC to 80ºC with 3ºC intervals. The thermophilic enzymes were the least active at every temperature, while the psychrotrophic variant was tenfold as active with the mesophilic one between these two. The temperature dependences of the reaction rate constants showed deviations from the linear Arrhenius-relationship. The nonlinear Arrhenius-plots points to a structural change at a distinct temperature.

Hydrogen isotope exchange studies offer a powerful tool to compare the conformational flexibilities of protein molecules. Our measurements indicate, that while thermophilic enzymes (IPMDH, GAPDH) are much more rigid than their mesophilic counterparts at room temperature, but at their respective optimum temperatures the global flexibilities of the two enzymes are almost identical. This phenomenon shows that adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (H/D exchange) and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25°C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well, however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature. The flexibility patterns of all three IPMDHs shows some deviation near the temperature where the break in the Arrhenius-plot appears, which indicates that the change in the activity is in close relationship to the change in conformational flexibility.

Computational design of proteins to a particular function is a very promising area of biotechnology. The first part of the work is to understand the principles of nature, hence molecular dynamics simulation are being carried out to underlie the in vitro results.

In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic counterparts. From the atomic coordinates several structural parameters were calculated, compared and evaluated using statistical methods. We concluded that different protein families adapt to higher temperatures by different sets of structural devices (Figure 2). The only generally observed rule is an increase in the number of ion pairs with increasing growth temperature, other parameters just show a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways than moderately thermophilic ones. The preferences of these two groups are different with regard to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.

We carry on with the statistical comparison of mesophilic and thermophilic enzyme pair, with special emphasis on the global conformational flexibilities.

The functional consequences of conformational flexibility at the active site are studied by observing the temperature dependence of the kinetic isotope effect, to reveal the mechanism of quantummechanical channeling in the case of hydride transfer enzymes.

In our research we combine molecular modelling, tools of recombinant DNA techniques, and a variety of physical methods, which includes differential scanning calorimetry (DSC), circular dichroism spectroscopy (CD), hydrogen-deuterium exchange, Fourier transformed infrared spectroscopy (FTIR), fluorescence and ultraviolet spectrophotometry (UV), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and analytical ultracentrifugation.