Analytical chemistry at the extremes: Ultrafast and single molecule fluorescence spectroscopic investigations of biological systems

This dissertation describes bioanalytical research at two extremes, the ultra-fast timescale and single-molecule spectroscopy. Ultra-fast spectroscopy was used to characterize the spectroscopic and photophysical behavior of a fluorescent analogue of proline, indoline-2-carboxylic acid (I2CA). Single molecule spectroscopy was used to monitor the Ca2+ and target dependent conformation changes of the calcium signaling protein, calmodulin (CaM) by the incorporation of an energy transfer fluorescent dye pair on CaM.; CaM, containing two genetically engineered cysteine residues, was labeled with a single donor and single acceptor dye. The simultaneous labeling of CaM required the development of an HPLC method to isolate the CaM molecules containing only a donor and acceptor dye (CaM-DA). CaM-DA functions as a sensor of peptide and drug molecule binding and was used to examine the target binding behavior of CaM. Surface immobilization was shown to perturb with the target binding functionality of CaM-DA, therefore, a construct was designed where CaM was fused to maltose binding protein (MBP-CAM). This fusion allows the immobilization of BP-CaM in low weight-percent agarose gels. In these gels, the Ca2+ and target binding conformational changes of CaM were assayed using MBP-CaM labeled with donor and acceptor dyes, MBP-CaM-DA. Experiments reveal that MBP-CaM-DA binds to the CaM antagonist, amitriptyline, with the same affinity both free in solution and in agarose gels. Additionally, MBP-Cam-DA, immobilized in agarose gels, was used to measure the high affinity binding constant of a peptide target of CaM directly. Single molecule fluorescence spectroscopy was used to monitor the conformational fluctuations of CaM on both the micro- and millisecond timescales. These experiments show differences in the dynamics of CaM in the presence and absence of calcium.; Lastly, a thorough investigation of the pH dependent photophysics of indoline and indoline-2-carboxylic acid (I2CA) using ultra-fast spectroscopy is presented. I2CA is a fluorescent analog of proline where its emission dipole is tethered to the peptide backbone; thus eliminating floppy side chain motions present in the native fluorescent amino acids. I2CA allows the global conformational changes of enkephlin-like penta-peptides to be examined. This work determined the relaxation mechanisms of indoline and I2CA by comparison to other aromatic amines like indole.