RESEARCH:

Our research interests are in the fields of cell biology, protein chemistry and enzymology with focus on structure-function relationships. All the projects have cold-adaptation as a backdrop. Firstly, we would like to understand better how enzymes can work at near freezing temperatures in various organisms (cold-adaptation of enzymes). This we do by the rational design of enzyme variants linked with molecular dynamics simulations. Secondly, we have directed attention to membrane proteins and lipid rafts. We want to see if rafts are present in cold-adapted fish. We want to characterize their lipid and protein compositions as well as define structural and functional interactions of their molecules. Finally, the promiscuity of enzymes is of interest to us. We want to wake up and/or enhance dormant activities guided by computational predictions. The experimental organism are either Atlantic cod or a Vibrio splendidus marine bacterium, and the enzyme we most often work with is alkaline phosphatase.

Current projects:

(1) Alkaline phosphatase is an enzyme with a wide distribution in the biosphere, yet its role is unknown in many instances. In animals, the enzyme is bound on the outside of cell membranes with a phosphatidylinositolglycan (GPI) anchor. The enzyme is a glycoprotein but the benefit of the sugar chains is unknown. GPI-anchored enzymes are associated with lipid-rafts, a specific collection of certain types of lipid molecules and proteins that can be isolated as detergent resistant membrane parts. We have already characterized the lipid and fatty acid composition of the intestinal epithelium cells, both the brush border and basolateral parts from Atlantic cod (Gadus morhua). We intend to isolate the phosphatase from the brush border membrane with its associated membrane molecules and analyse those in the hope of gaining a better idea regarding its role.  We also intend to produce the non-glycosylated enzyme and measure if the sugars have an effect on basic properties of the enzyme. This project involves the isolation and characterization of proteins and lipids. Mass spectrometry will be employed in the proteomics work and sequencing of glycans.

(2) The dynamic movements that are inevitable within protein structures due to their inherent flexibilities shape enzyme function and specificity. Our case study of the Vibrio alkaline phosphatase (VAP) by molecular dynamics (MD) revealed some of the underlying networks of intramolecular interactions and communicating residues within the structure. A pattern of asymmetric flexibility was discovered in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra- and intermolecular interactions. Those results provided a structural rationale to the half-of-site mechanism previously proposed. We want to further understand how this can influence protein function and stability (at low temperature) and modulate conformational changes and allostery. This project involves the generation of enzymes with altered primary structure by mutagenesis, and elucidation of their tertiary structure by homology modelling or x-ray crystallography. Both kinetic and physical measurements are performed from the perspective of stability and structural molecular dynamics at various temperatures using fluorometry, circular dichroism, differential scanning calorimetry/isothermal titration calorimetry, electron magnetic spin resonance, metal content analysis etc.  Computer calculations (MD) are used to guide in the design and interpretation of experiments.

(3) Promiscuous activities in enzymes are predicted to be an ancient relic from the early period of life an Earth when enzymes were few and with broad specificity. We have discovered the inhibition of a cold-active Vibrio alkaline phosphatase (VAP) by imipenem but not by other APs tested from E. coli or shrimp). This was predicted by in silico modelling of metallo-β-lactamase active sites. We demonstrated that the inhibition was specific to imipenem, and not seen with ertapenem, meropenem, ampicillin or penicillin G. We are using computer modelling (in silico analysis) to detect homologous scaffolds where both the spatial and electrostatic congruence of the active sites are considered in order to predict replacements of residues that might bring back new activities, such as sulfatase activity and general β-lactamase activity.

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