• to find how the combination of structural and dynamic attributes are optimized to ensure stability and activity at low temperatures.

• to measure the local dynamic nature of cold-active protein structures in order to understand the delicate mechanism responsible for the balance between rigidity, molecular fluctuations, and catalytic function.

• to asses strategies for improving heat stability with high activity for applications in biocatalysis and biotechnology.

Previous and current research

The Earth's biosphere is dominated by cold habitats, and therefore, cold-adaptation of enzymes is, the norm rather than the exception.  Most such enzymes meet the challenge to drive reactions forward environments of low thermal by attaining the required dynamic catalytic mobility.  As the temperature increases, these enzymes commonly suffer temperature denaturation, since evolutionary pressure has not directed residue selection to deal with temperature stability. 

Several factors contribute to protein stability.  In addition to hydrophobic interactions, non-covalent interactions such as hydrogen bonds, van der Waals interactions, and ion-pair networks (salt-bridges) can provide all of the stabilizing energy needed.  Disulfide bonds are the covalent bonds that proteins can further utilized for stability, and they do contribute significantly to stability of several proteins.   Although the introduction of new disulfide bridges by genetic engineering often leads to the expected stabilization, many mutants show no effect, or even destabilization, when compared with the wild-type enzyme.  Possible reasons for detrimental effects is when the introduction of a disulfide bond introduces strain, because the required stereochemistry is not exact, or because substituting a residues with cysteine looses some favorable interactions, or causes steric contact strain [see: B. Asgeirsson, B.V. Adalbjornsson and G.A. Gylfason, Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1774 (2007) 679-687]. 

            Vibrio sp. alkaline phosphatase (AP) is cold-active and particularly heat-intolerant [see: J.B. Hauksson, Ó.S. Andrésson and B. Ásgeirsson, Heat-labile bacterial alkaline phosphatase from a marine Vibrio sp., Enz. Microbial. Technol. 27 (2000) 66-73.].  Cold-adaptation of enzymes is believed to involve less internal adhesion in the proteins structure in order to allow more dynamic movement within the active site.  It is not always clear if the critical flexibility is global or local to the active site, and direct measurements of this elusive concept is notoriously difficult, but can be approached by H/D exchange experiments or electron spin resonance. 

In our present study we use protein engineering to introduce new cysteine into the cold-active Vibrio alkaline phosphatase.  The aim is to place an electron paramagnetic resonance spin-probe in various locations and test if enzyme activity is indeed linked with polypeptide chain mobility. This would be expected if dynamic movement within the conformational ensemble of the enzyme is a determinant. Several mutations that are likely to affect catalysis are being compared.