Master thesis by Salome Veshaguri – Niels Bohr Institute - University of Copenhagen

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Master thesis by Salome Veshaguri

Enzymes are dynamic entities adopting a wide landscape of conformations interconverting on wide range of timescales. The prevailing hypothesis on enzyme dynamics assumes conformational states to be directly related to activity states 1,2.

Nevertheless, this has never been experimentally validated because the technique to observe single enzyme functioning on atomic resolution and on physiologically relevant timescales still remains to be achieved. In this thesis to provide a link between conformational and activity states of enzymes we employed a recently developed single molecule setup based on TIRF microscopy. The method is capable of studying activities of single enzymes immobilized on liposomes in a high throughput manner and with relative high temporal resolution3-6.

Screening the activities of naturally glycosylated TLL (gTLL) and the non-glycosylated mutant (ngTLL) allowed us to document for the first time existence of long-lived activity states. The high quality data permitted us to plot functional landscapes for the two enzyme variants that showed an optimal activity state that the enzyme spends majority of its time. Furthermore ngTLL was found to exhibit increased number of transitions compared to gTLL. To identify if functional landscapes are correlated with conformational landscapes we examined the stability of two variants. By denaturation experiments we found ngTLL to be less rigid. Indicating that it undergoes new conformational states more often.

Our finding show more flexible enzyme also has an increased number of activity states and thus provide a link between conformational and activity state.

Master thesis Defence by Salome Veshaguri