Single-Molecule and Ensemble Measurements Inform on the Role of Protein Crowding at Interfaces

Surface-adsorbed proteins can critically affect the efficacy of materials designed for biological applications. For example, physiologically relevant quantities of serum albumin stabilize nanoparticles in solution, while decreased protein concentrations induce nanoparticle aggregation. The first aim of this dissertation was to characterize the crowding mechanism of the most abundant serum protein, serum albumin. Many studies have explored serum protein conformation on albumin-crowded surfaces, but unanswered questions about the mechanism of interaction between different proteins remain. Single-molecule high-resolution imaging with photobleaching (SHRImP) was applied to measure dye-conjugated fibronectin’s unfolding in varying conditions of crowding with serum albumin on aminosilanized glass. Our results indicated that serum albumin sterically restricts fibronectin’s unfolding during the adsorption process. Because fibronectin’s conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity. The second aim of this dissertation was to characterize the chiral aggregation of different protein-nanoparticle aggregates as a function of crowding and to gain insight into the protein-nanoparticle aggregation mechanism. Serum albumin induces gold nanorods to aggregate and produces protein-nanorod aggregates that exhibit circular dichroism. These aggregates could be applicable to sensitive chirality detection, but the mechanisms by which they form and produce circular dichroism are not well understood. Circular dichroism spectroscopy and scanning electron microscopy were implemented to characterize the nanorod aggregates produced with globular and fibrous proteins over varied protein/nanorod ratios. Serum albumin and other globular proteins produced similar chiral protein-nanorod aggregates that were distinct from the aggregates produced by fibrous proteins. Further, CTAB, a common nanorod stabilizing agent, enhanced or inhibited protein-nanorod chiral aggregation depending on its absolute concentration, which provided insight on the importance of protein-protein interactions in protein-AuNR aggregation. Variations in aggregation patterns elicited by different proteins could be applicable for chiral nanostructure engineering and biosensing.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Natural Sciences

Grantor

Rice University

Year dc:date.issued

2021

Author dc:creator

• Warning, Lauren Amanda

Advisor dc:contributor.advisor

• Link, Stephan

• single-molecule microscopy
• chiroptical sensing
• protein corona

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/111224

OAI identifier oai:identifier

oai:repository.rice.edu:1911/111224