BINDING AFFINITIES OF FLAVINS TO RIBOFLAVIN BINDING PROTEIN USING FLUORESCENCE SPECTROMETRY AND ISOTHERMAL TITRATION CALORIMETRY, AND ESTIMATED BINDING ENERGIES USING COMPUTATIONAL APPROACHES
Open Access
- Author:
- Yazgi, Habib A
- Area of Honors:
- Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Julie B Ealy, Thesis Supervisor
Dr. Sandy Feinstein, Thesis Honors Advisor - Keywords:
- Riboflavin binding protein
Riboflavin
Cancer
Folate receptor alpha
Binding energy
Binding affinity
Isothermal Titration Calorimetry
Fluorescence Spectroscopy
ICM-Pro - Abstract:
- Riboflavin binding protein is specific for transporting riboflavin from the human mother to the fetus during pregnancy. This protein is a highly conserved protein and is found in different species, which supports its importance in sustaining the development of the fetus. Elevation of riboflavin binding protein (RBP) has been associated with breast cancer in women. This elevation suggests that it might be a good biomarker to detect early breast cancer. Recently, riboflavin binding protein (RBP) antisera has been observed to cause riboflavin depletion in the human cervical cancer cell line (HeLa). This observation suggests that RBP can be a point of intervention for therapeutic drugs against cancer. Modern drug development techniques are attempting to rely more on computational approaches to measure the binding affinity of ligand-protein interactions and these approaches will save time and money. However, computational programs do not always account for all the ligand-protein interactions that contribute to binding affinity. This failure can contribute to discrepancies between wet bench lab techniques and a computational approach. Thus, we assessed how ICM-Pro (computational software) and fluorescence spectrometry performed in the determination of the binding affinity of riboflavin, lumichrome, FMN, and FAD. These results were compared to the established method (isothermal calorimetry titration (ITC)). ITC and fluorescence spectrometry used RBP, but ICM-Pro used human folate receptor alpha (4LRH.pdb), which is an evolutionarily related protein to RBP. Human folate receptor alpha protein has also been implicated in cancer, and researchers have been striving to develop new cancer drugs for this protein. For that reason, we also have compared 4LRH.pdb to RBP sequences and structures to each other. These results will potentially provide novel insights into these two proteins that can be utilized for potential cancer drug development for RBP as well as human folate receptor alpha. We have found that ICM-Pro results did not match the results from ITC, perhaps because two different proteins were used. However, future work is needed to verify these results. Fluorescence spectrometry results had consistent difference values compared to the ITC results. These differences suggest that the less expensive fluorescence spectrometry could be used to measure accurate binding affinities. However, testing more flavins is needed to verify the consistency of these differences between fluorescence spectrometry and ITC. Structural analysis of RBP and human folate receptor alpha demonstrated a significant similarity between the two proteins thus indicating their similar functions. Riboflavin was shown to have almost identical positions in both proteins with respect to Tyr75 and Trp156 in RBP and their corresponding amino acids Tyr85 and Trp171, respectively, in human folate. The distance between riboflavin and the two amino acids was almost identical in both proteins. The structural analysis of the position of riboflavin in 4LRH.pdb and RBP and the measurement of the flavins’ binding affinities using ITC, ICM-Pro and fluorescence spectrometry provide information on the similarities and differences between these two proteins. This research also provides avenues for further structural investigation of drug-like and nondrug-like flavin molecules in 4LRH.pdb and their relationship to the estimated binding energies using ICM-Pro. This will further enhance our understanding of the relationship between RBP and human folate receptor alpha and provide new opportunities for future cancer drug development.