THE EFFECT OF SOLVENT ON SILICON AND GERMANIUM RHODAMINES AND SYNTHESIS OF THE AQP4EX C-TERMINUS EXTENSION PEPTIDE

Faraz Abounorinejad

doi:10.7273/000006904

SECTION ONE: FLUORESCENCE EXPERIMENTS Red-emitting group XIV rhodamines have emerged as promising fluorescent probes for biophysical experiments, especially on the single-molecule level. This is due to their high fluorescence quantum yields, resistance to photobleaching, and high solubility in water. These fluorescent probes are ideal for biological experiments due to their near-infrared excitation range, which is where biological samples are mostly transparent. While studying the photophysical parameters of two of these probes, a silicon and a germanium rhodamine zwitterion, our group noticed differences in the behavior of these molecules based on the solvent they are dissolved in. These prompted further experiments to investigate the change in spectra, change in fluorescence quantum yields, and change in fluorescence lifetimes in solvents with differing pH or polarity. pH-dependent experiments revealed that the silicon and germanium rhodamines studied are mostly unaltered in pH 4 to pH 10 buffers. Polarity dependent experiments revealed that the silicon and germanium rhodamines experience a solvatochromic red shift in solvents with lower polarity and experience a significantly heightened fluorescence quantum yield in a 4:1 ethanol:methanol solvent. Fluorescence experiments at a temperature of 77K revealed that their spectra are affected by the hydrogen bonding capability of the solvent. This finding is consistent with the measured changes in Stokes Shift based on solvent used. This thesis presents these findings along with a partial photophysical characterization of these molecules at room temperature and 77K. SECTION TWO: PEPTIDE SYNTHESIS FOR AQP4 STUDIES Human aquaporin 4 (AQP4) is the primary water channel in the central nervous system (CNS). It functions to maintain water-ion homeostasis, and the dysregulation and altered expression of it is a part of the pathology of conditions such as neuromyelitis optica (NMO), brain ischemia, and brain tumors. Due to its potential impact on human health, AQP4 has been studied extensively to understand the mechanisms through which it is controlled, especially in regard to its localization at the astrocyte perivascular endfeet processes near the blood brain barrier. A key interaction in this regard is between AQP4 and α-syntrophin, a protein that coordinates it to this location in the cell. Recent studies have shown that an isoform of AQP4 with an extended C-terminus, AQP4ex, is critical for this interaction. Though there is strong evidence this interaction takes place, a direct interaction between AQP4 and α-syntrophin has yet to be observed. Additionally, the mechanism for the interaction is unknown and it is not clear what role the canonical C-terminus or extended C-terminus may play. Because of this, our group has begun setting the groundwork for experiments to observe this interaction. As a part of this, we have synthesized three peptides to represent the C-terminus extension in such an experiment, two that are the full extension and differ only in the inclusion of a cysteine, and one that includes only the last 7 amino acids of the extension. The process of synthesizing, purifying, and identifying these peptides is presented in this thesis.

THE EFFECT OF SOLVENT ON SILICON AND GERMANIUM RHODAMINES AND SYNTHESIS OF THE AQP4EX C-TERMINUS EXTENSION PEPTIDE

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