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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorCramer, Steven M.
dc.contributorBelfort, Georges
dc.contributorBarquera, Blanca L.
dc.contributorMcCallum, Scott A.
dc.contributorGarcía, Angel E.
dc.contributorKane, Ravi S.
dc.contributor.authorSrinivasan, Kartik
dc.date.accessioned2021-11-03T08:18:55Z
dc.date.available2021-11-03T08:18:55Z
dc.date.created2015-03-09T11:52:33Z
dc.date.issued2014-12
dc.identifier.urihttps://hdl.handle.net/20.500.13015/1328
dc.descriptionDecember 2014
dc.descriptionSchool of Engineering
dc.description.abstractThe 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.
dc.description.abstractFurther, to probe the energetics of face specific binding of ubiquitin to SAMs containing a MM cation exchange ligand single molecule force spectroscopy (SMFS) was employed. Cysteine mutations were performed at two different strategic locations on the protein surface to exert control over the orientation of the covalently immobilized protein. The exposed face of the protein was interrogated by performing force spectroscopy measurements using a probe functionalized with a SAM of the MM cation exchange ligand, "Capto ligand". The force spectroscopy results indicated that the binding face identified by NMR for interaction with this MM surface indeed show a significantly high value of unbinding free energy as compared to measurements with the non-binding face. The use of SMFS establishes a potential link between experiments and Molecular dynamics (MD) simulations through the determination of single molecule "face specific" binding energetics. Finally, the insights gained from this fundamental work on protein-MM surface interactions would be employed in concert with MD simulations in future to create predictive models for protein binding in order to improve the separation capabilities of industrial process mixtures.
dc.description.abstractFurther, quartz crystal microbalance with dissipation (QCM-D) was employed to obtain real time mass adsorption and desorption data of proteins binding to SAMs of MM ligands that mimic a chromatographic resin surface, in a continuous flow mode. A systematic approach was employed to investigate effects of ligand density by creating SAMs of different MM ligand densities on gold coated quartz crystals. In the present study, two geometrically different MM cation exchange ligand systems were employed to delve into the differences in protein binding kinetics. A rigorous analysis was performed to quantitatively probe protein adsorption and desorption kinetics under the different conditions employed in this study. Moreover, binding behavior of proteins under different fluid phase conditions was also examined in order to delineate the fundamental reasons for protein selectivity.
dc.description.abstractRecent advances in the development of multimodal (MM) chromatographic resin systems, have produced materials that create exclusive windows of selectivity as compared to traditional single mode chromatographic systems. The presence of multiple interaction modes combined with the targeted use of specific mobile phase conditions, creates unique protein binding selectivities which were previously unachievable using single interaction modes. Although MM resins offer significant advantages for bioseparations, there is a lack of fundamental understanding of the nature of binding of proteins to chromatographic ligand surfaces. This project combines the use of various high resolution experimental approaches to establish a deeper molecular level understanding of the processes that govern protein binding and selectivity in MM chromatographic systems.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectChemical engineering
dc.titleBiophysical investigation of protein-ligand interactions in multimodal chromatography
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid174862
dc.digitool.pid174863
dc.digitool.pid174864
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Chemical and Biological Engineering


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