Date of Award

2016

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Administrative Home Department

Department of Chemistry

Advisor 1

Ashutosh Tiwari

Committee Member 1

Haiying Liu

Committee Member 2

Martin Thompson

Committee Member 3

Caryn Heldt

Abstract

Surface hydrophobic interactions in proteins play a critical role in molecular recognition, influence biological functions, and play a central role in many protein misfolding diseases. As significance of surface hydrophobic interactions in age-related proteinopathies is becoming clear; it has led to an increased demand for better probes and tools to sense and characterize protein surface hydrophobicity. Current commercially available fluorescent probes such as 8-anilino-1-naphthalene sulfonic acid (ANS), 4,4′ -dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS), 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN), tetraphenylethene derivative, and Nile Red can sense proteins average hydrophobicity. However, probe limitations prevents their application for measuring the protein surface hydrophobicity. Some of the major deficiencies of these fluorescent probes are: poor solubility in water, overestimation of fluorescence signal due to contribution from hydrophobic as well as electrostatic interactions, and weak signal when bound to solvent exposed hydrophobic surface of proteins due to quenching. As a consequence of these limitations the above fluorescent dyes do not provide accurate measure of proteins surface hydrophobicity. Therefore, in this study we focused on designing and testing novel fluorescent probes for selectively reporting the surface hydrophobicity of proteins. For the first project, we chose 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) based fluorescent probes as these are highly fluorescent in both non-polar as well polar media. To increase water solubility we substituted 2-methoxyethylamine group at 3,5-position of the BODIPY core. For increasing hydrophobic sensing we focused our efforts on substitutions at meso position on BODIPY dye. These BODIPY-based surface hydrophobic sensors (HPsensors) showed a much stronger signal compared to ANS, a commonly used hydrophobic probe. The probes showed a 10- to 60-fold increase in signal strength compared to ANS for the BSA protein. For the second project, we modified the commercially available ANS dye with a succinimide-functionalized ethynyl derivative that offers facile reaction with amine residues of proteins at physiological pH. This modification of ANS with a reactive NHS ester favors crosslinking of the dye on proteins surface with lysine or arginine residue present near surface hydrophobic regions. SDS-PAGE results show that the dye is covalently linked to the proteins. To map the hydrophobic surface on proteins, covalently modified proteins will be digested and analyzed using mass spectrometry. Following that, the proteins hydrophobic surface will be visualized using crystallographic structure database for in-silico screening of small molecule libraries. These small molecules will be tailored to fit the exposed hydrophobic surface by rational drug design approach and explored for novel therapeutic avenues.

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