Biophysical investigation of protein-ligand interactions in multimodal chromatography
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Authors
Srinivasan, Kartik
Issue Date
2014-12
Type
Electronic thesis
Thesis
Thesis
Language
ENG
Keywords
Chemical engineering
Alternative Title
Abstract
The chromatographic resin surfaces were mimicked by the use of self-assembled monolayers (SAMs) functionalized with specific MM ligand chemistries. The binding process of proteins to these fabricated MM surfaces was investigated from a molecular, thermodynamic and kinetic stand point. A nanoparticle system was employed that can simulate a chromatographic resin surface while also being amenable to isothermal titration calorimetry (ITC) and solution NMR. NMR titration experiments were carried out with 15N labeled ubiquitin to investigate the interactions of ubiquitin with nanoparticles functionalized with two industrially important multimodal ligands. The ITC results suggested that binding to both multimodal ligand surfaces was entropically driven over a range of temperatures and that this was due primarily to the release of surface bound waters. In order to reveal structural details of the interaction process, binding-induced chemical shift changes obtained from the NMR experiments were employed to obtain dissociation constants of individual amino acid residues on the protein surface. The residue level information obtained from NMR was then used to identify a preferred binding face on ubiquitin for interaction to both multimodal ligand surfaces employed in this study. In addition, Electrostatic Potential and Spatial Aggregation Propensity maps were used to determine important protein surface property data that are shown to correlate well with the molecular level information obtained from NMR.
Description
December 2014
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY