engleski

Computational Study as Guideline for Experimental Research

Nowadays millions of tons of inadequately or insufficiently treated sewage, industrial and agricultural waste are released directly into the groundwater, rivers, lakes, seas and oceans, thus introducing numerous species of pathogenic agents. This contamination affects our natural ecosystems and directly affects different food and beverage manufacturing process. Therefore, detection of pathogens in water must meet specific quality requirements. The current procedures relay on time consuming bacterial culture plating methods or (to a much smaller degree) using expensive molecular methods such as Enzyme Linked Immunosorbent Assays (ELISA), reporter enzyme-dependent detection and Polymerase Chain Reaction (PCR). The main goal of the presented research is to contribute to the development of a novel pathogen detection assay for monitoring water safety that relays on horseradish peroxidase (HRP), an enzyme that was shown to have a great potential in development of novel protein-fragment complementation assays [1]. In order to achieve this goal, a combined research consisting of experimental and computational methods is conducted. The role of computational methods is to provide a deeper understanding of experimental data and to enable insight into the molecular level of the systems of interest. Results of computational simulations pointed to the importance of glycosylation on structural and dynamical properties of HRP which affect its overall stability. Particularly interested is propagated effect of glycosylation on electrostatic properties of the inner core of the enzyme [2]. These results helped resolving experimental issues. Further, experimental attempts to attach the single strained DNA oligonucleotide to the HRP, which is necessary for the development of proposed pathogen detection assay, were supported by computational simulations. Availability of different enzyme functional groups for the oligonucleotide attachment were studied by molecular dynamics simulations. Results of simulations helped experimental attachment of the oligonucleotide and showed that it should not have significant influence on enzymatic activity which was experimentally confirmed.

molecular dynamics, horseradish peroxidase, glycosylation, protein-oligonucleotide

nije evidentirano