Single molecule dynamic force spectroscopy of alginate-epimerase interactions using optical tweezers
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- Institutt for fysikk 
Alginate, a naturally occurring polysaccharide found in various cell lines, is synthesized as a homopolymer polymannuronic acid also known as mannuronan or PolyM. Conversion of the polymers subunits β-D-mannuronic acid (M) to the C-5 epimer α-L- guluronic acid (G) residues occurs during a post-polymer epimerization processes catalyzed by enzymes known as the mannuronan C-5 epimerases. The aim of this study has been to further explore the underlying nature of the epimerases, some of which are thought to create G-subunits by moving processively along the alginate chains. The smallest epimerase from the mannuronan C-5 genes, know to form polyalternating alginate structures (PolyMG), AlgE4, as well as AlgE6, which manly introduce long G-block stretches, were further investigated.Moreover, two synthetically created epimerases, AlgE46 and AlgE64, were provided by the department of Biotechnology, NTNU. Investigation of the synthetic epimerases may provide insight into the importance of the structural composition of the mannuronan C-5 epimerases.Dynamic force spectroscopy of single-molecule measurements between the substrate chains PolyM and PolyMG, and the epimerases AlgE4, AlgE6, AlgE46 and AlgE64 were carried out using the optical tweezers.Binding strengths ranging from 1.5 − 14 pN and 4 − 15 pN at loading rate intervals 6−450 pN/s and 50−1200 pN/s were measured for the AlgE4-mannuronan and the AlgE4-PolyMG complex, respectively. For the AlgE6-mannuronan and the AlgE6-PolyMG complex, on the other hand, the measured binding strengths ranged from 2.5−7.5 pN and 4.5−7.5 pN at loading rate intervals 13−340 pN/s and 70 − 430 pN/s, respectively. Finally, for the synthesized epimerases, binding strengths ranging from 2 − 6.5 pN, 4 − 9.5 pN and 3 − 8 pN at loading rate intervals 10 − 450 pN/s, 10 − 240 pN/s and 50 − 300 pN/s were measured for the AlgE46-mannuronan, AlgE64-mannuronan and AlgE64-PolyMG, respectively. All epimerases, whether naturally occurring or synthetically created, were shown tobind stronger to PolyM than to PolyMG.Furthermore, single energy barriers were located between 0.6 − 1.35 nm for all investigated complexes. The interplay of non-covalent interactions between the complexes (indicated by the location of the energy barriers), as well as the bond lifetimes were consistently greater for the complexes involving PolyM, than for the ones involving PolyMG.Although certain tendencies were observed when comparing naturally occurring epimerases with the synthesized ones, no ﬁrm conclusion could be drawn to the underlying nature of the diﬀerent structural components of the mannuronan C-5 epimerases. However, ongoing NMR-spectroscopy studies on AlgE64 and AlgE46 may provide more information concerning the epimerization process of mannuronan/polyalternating alginates, such that the obtained observation from this study can be put in a biological context.Lastly, the AlgE4 epimerase was concluded to processively epimerize mannuronan to form polyalternating alginate chains. Similar conclusions for the other epimerases could not be drawn since information regarding the catalytic constants of the epimerization process of a single M-residue for these epimerases are not accessible in the litterature.