Bachelor thesis defense by Stine Eiersholt – Niels Bohr Institute - University of Copenhagen

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Bachelor thesis defense by Stine Eiersholt

FRET assay to probe Cytochrome P450 Reductase domain movements

Cytochrome P450 Reductase (POR) is a crucial redox enzyme acting as a molecular hub activating all microsomal Cytochrome P450s and controls a plethora of metabolic cascades. POR contains two domains, which binds the FAD and FMN cofactors, through which electron transfer occurs. POR is believed to have complex structural dynamics that are expected to contribute to its catalysis by controlling and limiting the electron transfer. However, to date, there is no reliable assay to probe POR dynamics and conformational equilibrium. To address this issue I have developed an assay FRET assay that acting as a spectrometric ruler, can measure the conformational equilibrium distance of POR. Two double cysteine mutants were chosen, so that the open and closed distances of POR were in the FRET range, and were consequently labeled with Cy3 and Cy5 maleimide fluorophores. My measurements reveal the existence of FRET in POR To further confirm the assay I decided to use already established regulatory inputs (ligands and ionic strength variations) to shift the conformational equilibrium and quantify the associated FRET changes. My findings confirm the existences of a conformational equilibrium of POR that can be furthermore shifted by regulatory inputs. I expect that further experiments with FRET will help decipher the roles of equilibrium states and subsequently show how these states influence the dynamics. Finally I anticipate that this method with the chosen mutants used in single molecule studies will provide key structure-function correlations.