Establishing Forster Resonance Energy Transfer Protocol for Extracellular Matrix Proteins – UROP Summer Symposium 2021

Establishing Forster Resonance Energy Transfer Protocol for Extracellular Matrix Proteins

Bianca Hernandez

Bianca Hernandez

Pronouns: She/Her/Hers

UROP Fellowship: Engineering
Research Mentor(s): Do Hoon Kim, PhD Candidate
Research Mentor Institution/Department: College of Engineering, Department of Chemical Engineering

Presentation Date: Wednesday, August 4th
Session: Session 2 (4pm-4:50pm EDT)
Breakout Room: Room 1
Presenter: 4

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Understanding protein interactions is important as it could serve as a basis for designing extracellular matrices (ECM) in-vitro to mimic in-vivo characteristics which would help elicit better biological responses from cells and be useful for disease modeling and drug testing. The purpose of this research is to create Forester Resonance Energy Transfer (FRET) technique for a protein called fibronectin to study its conformational changes and molecular interaction with other proteins. FRET is based on the distance between two fluorophores and their resonance frequency. When the donor fluorophore is excited, it emits energy that excites the acceptor fluorophore. Here, fluorophores are tagged to fibronectin at a specific binding sites and the energy transfer between donor and acceptor fluorophore is utilized to help us gauge their spatial distance defined by the conformation of fibronectin. To create the protocol, calibration curves of concentration vs absorbance were created for both Alexa Fluor 488 (AF488) and fibronectin (FN) using a spectrophotometer and Beer’s Law. Then FN and AF were conjugated together at a molar ratio of FN:AF at 10:1 and 30:1. Reaction solution was then dialyzed to remove any unbound dyes. By measuring the absorbance of the final solution we were able to calculate the concentration of AF and FN and create a calibration curve of FN & single Alexa Flour (AF488) tag. Utilizing these steps, AF488+FN conjugates can be tagged with another fluorophore, AF546 to create a dual-fluorophore fibronectin conjugate, which can be used to investigate the molecular conformation of fibronectin in presence of various proteins. Successful dual labeling of fibronectin is confirmed by creating a plot showing the changes in the intensity of acceptor fluorophore (AF #2) when placed in denaturing solution (guanidine chloride) of different concentration. Established protocol and calibration curves from this work will be used to confirm whether prepared samples are suitable for FRET studies, which in turn will provide insights on how proteins will behave and interact at a molecular level.

Authors: Bianca Hernandez, Do Hoon Kim, PhD Candidate
Research Method: Laboratory Research

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