Monomeric red fluorescent proteins (RFPs) are used extensively for applications in molecular biology research, and are especially suited for whole body imaging applications due to their longer excitation and emission wavelengths, which are less damaging and penetrate deeper into animal tissue. However, these proteins suffer from reduced brightness compared to other fluorescent proteins, and require further engineering, which is often achieved through random methods, incurring large time and resource costs. Here we propose a rational design approach to improve the quantum yield of RFPs by reducing conformational variability of the chromophore. We engineered mRojoA, a mutant containing a π-stack involving Tyr197 and the chromophore phenolate, to include the P63F/H/Y mutations on its other side, by simultaneously mutating neighbouring positions 16, 143, and 163. The brightest mutants that we found in each library, mRojo-VYGV, mRojo-VFAV, and mRojo-VHSV, exhibited 1.8- to 2.4-fold increases in brightness, and quantum yield increases of up to 2.1-fold. In all three mutants, the increases in brightness were predominantly due to improvements in the quantum yield and not the extinction coefficient. Solving the crystal structures of two of these mutants along with a dim variant allowed us to strongly infer a link between rigidity of the chromophore and increased quantum yield. In addition, back-mutating position 63 in the highest quantum yield mutant, mRojo-VYGV, reversed the improvement in quantum yield, indicating that Y63 was the primary residue responsible for the improved brightness of the protein. Unfortunately, the mCherry-VYGV mutant did not achieve a similar increase in quantum yield or brightness. This is likely due to the lack of a second bulky aromatic residue at position 197, which is present in mRojoA. Nevertheless, this rational approach could be applied to some other RFPs whose chromophores exhibit increased conformational variability in order to further improve their brightness.

A new synthetic methodology for the combinatorial preparation of C-terminus-modified Green and Red Fluorescent Protein chromophores is described. This method involves the modification of the previously reported [2+3] cycloaddition reaction scheme to incorporate new R2 groups in the imidate used in the final step. This is achieved through two primary routes: (a) the imidation of nitriles using hydrochloric acid gas and (b) the O-alkylation of amides using a variant of Meerwein's Salt to provide conjugated imidates. The preparation of fluorescent microcrystals and nanofibers from Green Fluorescent Protein chromophore derivatives via the reprecipitation method is also demonstrated. The properties of these microcrystals and nanofibers, especially in relation to the powder obtained from organic solvents, are also explored. Additionally, it is demonstrated that the size and shape of the microcrystals and nanofibers can be modulated with varying experimental conditions for RP. A new class of AIE-active GFP chromophores is reported. These chromophores contain a benzoxazole group on the phenyl ring and varying lengths of alkyl chains on the imidazolidinone nitrogen. These benzoxazole-based chromophores exhibit unique properties in the solid state not previously observed for GFP chromophore derivatives, namely, a broadening of the excitation spectrum and red-shifting of the emission, likely caused by excimer formation. The crystal structure also reveals a unique "hot-dog" stacking motif. Additionally, some projects which require further work are discussed at the end of the thesis. These include a stress-responsive GFP-based polymer and DNA-binding fluorophores.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.