Biophysical investigation of protein-ligand interactions in multimodal chromatography

Srinivasan, Kartik
Thumbnail Image
Other Contributors
Cramer, Steven M.
Belfort, Georges
Barquera, Blanca L.
McCallum, Scott A.
García, Angel E.
Kane, Ravi S.
Issue Date
Chemical engineering
Terms of Use
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
Full Citation
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.
December 2014
School of Engineering
Dept. of Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Troy, NY
Rensselaer Theses and Dissertations Online Collection
Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.