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Effective de novo design of proteins with targeted functions has potential in clinical, research, and industrial settings. As such, the ability to control the function of these de novo designed proteins is also of particular interest. While advancements have been made in the computational methods used to design proteins with desired structures, the biochemical basis for many desired functions is still unclear. One such function not currently well understood is the underlying causes for the differing brightnesses across the de novo designed mini fluorescence activating protein (mFAP) family. The mFAP’s bind and stabilize the GFP chromophore analog 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) to induce fluorescence. Previous research on 14 different mFAP proteins suggests that mFAP’s which hold the DFHBI dihedrals more planar tend to be brighter. In this research, the brightness and binding of the 14 mFAP’s used in previous studies as well as an additional 13 novel mFAP’s were characterized using a method developed for this research which allows for simultaneous determination of mFAP binding and relative brightness. This data was used to reexamine previous findings and investigate the efficacy of previously developed computational methods that identify mutations predicted to improve mFAP brightness. Characterization of these proteins also provided insight into the underlying factors of mFAP brightnesses. The computational methods which predict mutations suspected to improve brightness were found to be ineffective thus far. However, two factors, the root mean square (RMS) angle of the phenyl dihedral and the chemical shift position of the right peak in the 19F NMR spectra, strongly correlate with brightness suggesting that DFHBI dynamics and electronics both impact the brightness of mFAP bound DFHBI.

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