Synthesis and characterization of the structure of neutral flavin radicals in solution
Presentation Type
Abstract
Faculty Advisor
Johannes Schelvis
Access Type
Event
Start Date
25-4-2025 9:00 AM
End Date
25-4-2025 9:59 AM
Description
Cryptochromes and photolyases are very similar proteins that serve in biorhythm signaling and in DNA repair, respectively. These flavoproteins rely on the flavin adenine dinucleotide (FAD) chromophore to absorb light to carry out their function. For cryptochromes/photolyases, the FAD cofactor is in an stacked/folded conformation, and in all other flavoproteins it is in a unstacked/unfolded one, so the reason for this difference is of interest. FAD and flavin mononucleotide (FMN) exhibit structural differences that influence their behavior in solution. FAD contains an adenine ring that interacts with its isoalloxazine ring, leading to intramolecular stacking when dissolved in water. In contrast, FMN, which lacks an adenine ring, can’t exhibit such stacking. Upon dissolution in a water-methanol mixture, methanol disrupts the stacking interactions in FAD, leading to an unstacked conformation of FAD that behaves more similar to FMN. In this work, we study FAD unstacking in water and compare the results to FMN to obtain a better understanding of the role of stacked FAD in proteins. Fluorescence measurements confirmed that FAD fluoresces more intensely when destacked, with increasing methanol concentrations enhancing this effect. Resonance Raman spectroscopy was then employed to analyze how unstacking affected the FAD molecule, as this technique is sensitive to small structural differences. The ultimate goal is to find a marker signal that can be used to determine the level of stacking of FAD inside various proteins, and how this may affect the function of the protein.
Synthesis and characterization of the structure of neutral flavin radicals in solution
Cryptochromes and photolyases are very similar proteins that serve in biorhythm signaling and in DNA repair, respectively. These flavoproteins rely on the flavin adenine dinucleotide (FAD) chromophore to absorb light to carry out their function. For cryptochromes/photolyases, the FAD cofactor is in an stacked/folded conformation, and in all other flavoproteins it is in a unstacked/unfolded one, so the reason for this difference is of interest. FAD and flavin mononucleotide (FMN) exhibit structural differences that influence their behavior in solution. FAD contains an adenine ring that interacts with its isoalloxazine ring, leading to intramolecular stacking when dissolved in water. In contrast, FMN, which lacks an adenine ring, can’t exhibit such stacking. Upon dissolution in a water-methanol mixture, methanol disrupts the stacking interactions in FAD, leading to an unstacked conformation of FAD that behaves more similar to FMN. In this work, we study FAD unstacking in water and compare the results to FMN to obtain a better understanding of the role of stacked FAD in proteins. Fluorescence measurements confirmed that FAD fluoresces more intensely when destacked, with increasing methanol concentrations enhancing this effect. Resonance Raman spectroscopy was then employed to analyze how unstacking affected the FAD molecule, as this technique is sensitive to small structural differences. The ultimate goal is to find a marker signal that can be used to determine the level of stacking of FAD inside various proteins, and how this may affect the function of the protein.
Comments
Poster presentation at the 2025 Student Research Symposium.