The Development of Nontoxic, Sequence-Selective Small Molecule Bisboronic Acid Fluorescent Sensors for Proteins

Analysis of target proteins in live cells is fundamentally important to understanding their molecular roles in both normal biological processes and disease. While light microscopy and biochemistry have played fundamental roles in our understanding of cellular structure and function, the fact that cellular events often require proteins to fold, move, and assemble into specific macromolecular complexes has created a need for increasingly sophisticated methods to analyze these proteins in their native environments. Many of these methods rely on the ability to visualize and quantify proteins within the context of living cells or tissue using biological or chemical tags that can act as luminescent reporters.;Autofluorescent proteins such as GFP and a virtual rainbow of derivatives constitute the most widely used class of biological tags for live cell protein imaging. Labeling a protein by fusing it to GFP or an analog offers several key advantages, including genetic encodability, specificity, brightness, and myriad color options. Indeed, fluorescent proteins have enabled the direct visualization of protein localization, trafficking, turnover, "aging" (time since protein synthesis), and even interactions with other intracellular machinery. Despite these advantages, however, several limitations of this technology persist. The autofluorescent protein chromophore comes housed in a large (26 kDa) suitcase that can affect the physiological function of proteins fused to it. Autofluorescent proteins also have the potential to oligomerize, require anywhere from 40 minutes to 1 day to mature, exhibit significant pH sensitivity and cytotoxicity, and their fluorescence often can only be temporally controlled by harsh techniques such as photobleaching.;To overcome these limitations as well as supplement the set of tools available in the cell biologist's "fluorescent toolbox" attention has recently turned to small molecule labeling strategies that make use of an organic chromophore and a much smaller encodable peptide sequence. One class of reagents in this category includes pro-fluorescent biarsenical dyes such as FIAsH. ReAsH. CrAsH, and Cy3As. These cell permeable small molecules selectively label recombinant proteins tagged with a linear, or split tetracysteine motif via thiol-arsenic exchange reactions that convert the non- fluorescent 1,2-ethanedithiol-bound forms of these dyes into highly fluorescent protein- bound complexes on the order of minutes. This labeling strategy also affords temporal control, as the dye may be administered to cells at any time, and labeling reactions can be halted by treatment with excess thiol competitor. Despite their utility, however, biarsenicals are plagued by high background fluorescence and cytotoxicity, and can be challenging to apply in oxidizing cellular locales. Nontoxic, redox-insensitive alternatives that combine the brightness and small size of a biarsenical with the specificity and convenience of a fluorescent protein would thus be most valuable as bioorthogonal labeling strategies.;This doctoral thesis describes the development of a novel, nontoxic, small molecule fluorescent sensor that can function bioorthogonally with respect to other labeling strategies. Herein, a previously reported rhodamine-derived bisboronic acid sensor for saccharides, RhoBo, is shown to function as a turn-on fluorescent sensor for tetraserine motifs in engineered peptides and proteins. Through its dual arylboronic acid functionahties, RhoBo is able to selectively label an encodable, hairpin-prone tetraserine peptide sequence Ser-Ser-Pro-Gly-Ser-Ser (-SSPGSS-) with high affinity (452 nM) and a large fluorescent response (Deltaphi = 0.59). The response is selective for --SSPGSS- over other diserine and tetraserine peptide sequences, as well as other biologically relevant diols such as monosaccharides, disaccharides, and a microarray of naturally occurring mammalian cell glycans, and the response is consistent in the context of both short peptides and small proteins. RhoBo is also inherently cell permeable, able to traverse the saccharide-rich plasma membrane and gain access to the cytosol for intracellular protein imaging.;In an effort to achieve a more detailed understanding of the interactions between RhoBo and its tetrasesrine peptide ligand as well as acquire information that will be useful in the design of both higher affinity sequences for RhoBo and novel bisboronic acid-based fluorophores, a comprehensive spectroscopic, kinetic, and biophysical analysis of the RhoBo-peptide complex is also presented. To the best of the author's knowledge, RhoBo represents the first molecule in a new class of nontoxic, boronic acid- based fluorophores that are both peptide sequence-specific and have the potential to function bioorthogonally to other affinity labels.