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Stave 11 mutations alter the fluorescence of GFP

Amino acid changes to stave 11 are easy with 80 nt inserts.
-Alternating amino acid R groups in the GFP beta sheet face the protein interior.
-Those nearest the chromophore influence it the most.
-Synthetic DNA means any sequence is possible for stave 11.
Goal is to change the polarity of the chromophore environment.
-It is impossible to predict the exact effects on the chromophore.
-Think of the chromophore environment akin to a "solvent."
-Changing amino acids is like changing solvent (and the dielectric constant) near the chromophore.
It is crucial to recall that amino acid R groups point in specific directions.
-The overall protein structure ultimately determines these directions.
-The effect of a group's polarity is greatly enhanced by pointing it at a chromophore.
-X-ray structure data will ultimately help explain the various AA influences.
Fluorescence data is easy to obtain, and reveals key amino acids for stave 11 changes.
-The AA in stave 11 nearest the chromophore are Leu220, Glu222, and Val224.
-Changes to Glu222 and Val224 give fluorescent proteins.
-The spectra below show how V224 mutations influence the fluorescence.



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These results were presented at the Spring, 2020 ACS Meeting in Philadelphia,
https://doi.org/10.1021/scimeetings.0c02102.
Conclusions: The GFP chromophore exists as either the phenol (the A-state) or the phenoxide (the B-state, shown at right), and the ratio of the two is heavily influenced by the identity of the amino acid at position 224. Note that in the excitation spectra above, the emission arises from excitation of either the A-state at 395 nm, or the B-state at 475 nm, where the intensity if the two peaks reflects the abundance of the two species. These results prove that glutamic acid (E) stabilizes the phenol, while glutamine (Q) stabilizes the phenoxide. Also, the distance "d" in the diagram must be critical, since in V224N (N = asparagine), now the phenol is stabilized over the phenoxide (spectra not shown), where the only difference between Q and N is a single CH2 group. chromophore