Willson, Richard C.2019-09-172019-09-17August 2012014-08August 201Portions of this document appear in: Daniels, Charlisa R., Lydia Kisley, Hannah Kim, Wen‐Hsiang Chen, Mohan‐Vivekanandan Poongavanam, Carmen Reznik, Katerina Kourentzi, Richard C. Willson, and Christy F. Landes. "Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered‐charge peptide adsorbents." Journal of Molecular Recognition 25, no. 8 (2012): 435-442. And in: Kanakaraj, Indhu, Wen-Hsiang Chen, Mohan Poongavanam, Sagar Dhamane, Loren J. Stagg, John E. Ladbury, Katerina Kourentzi, Ulrich Strych, and Richard C. Willson. "Biophysical characterization of VEGF–aHt DNA aptamer interactions." International journal of biological macromolecules 57 (2013): 69-75. And in: Kisley, Lydia, Jixin Chen, Andrea P. Mansur, Bo Shuang, Katerina Kourentzi, Mohan-Vivekanandan Poongavanam, Wen-Hsiang Chen, Sagar Dhamane, Richard C. Willson, and Christy F. Landes. "Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations." Proceedings of the National Academy of Sciences 111, no. 6 (2014): 2075-2080. And in: Daniels, Charlisa R., Lydia Kisley, Hannah Kim, Wen‐Hsiang Chen, Mohan‐Vivekanandan Poongavanam, Carmen Reznik, Katerina Kourentzi, Richard C. Willson, and Christy F. Landes. "Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered‐charge peptide adsorbents." Journal of Molecular Recognition 25, no. 8 (2012): 435-442.https://hdl.handle.net/10657/4753Aptamers are single-stranded oligonucleotide (DNA or RNA) ligands that form three-dimensional structures and are capable of binding to pre-selected targets. In this work, we studied the dissociation kinetics and the effects of temperature and ionic strength on the D17.4 aptamer/IgE complex using fluorescence anisotropy, a homogenous, solution-phase technique. From the Arrhenius plot (ln(koff) vs. 1/T ), we calculated the dissociation activation energy (Ea) for the D17.4 aptamer/IgE complex, Ea= 16.0±1.9 kcal mol-1 at 50 mM NaCl, and 1 mM MgCl2. The role of nucleotides in the loop region of the D17.4 aptamer was investigated by mutating a single nucleotide in the loop region. To the best of our knowledge, this work is the first detailed experimental study of activation parameters for an aptamer/protein system. Separately, we employed single-molecule methods to investigate the complex underlying mechanisms of protein chromatography. Using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT), we present the direct mapping and kinetic characterization of individual functional sites on thin-film agarose ion-exchange matrices. By extracting single-protein adsorption and desorption kinetics at individual ligands, direct experimental evidence in support of the stochastic theory of chromatography is obtained. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five. Using the single molecule approach, we also investigated the influence of ionic strength on the heterogeneity of protein ion-exchange functional adsorption sites, and therefore the heterogeneity of elution profiles. We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand, and tuning of steric accessibility within the agarose support.application/pdfengThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).AptamersKineticsActivation parametersSingle-moleculeAdsorptionChromatography2019-09-17Thesisborn digital