Graduate Thesis Or Dissertation
 

Exploring the Limits of Ligation Rate and Specificity in Protein Immobilization Using the Genetically Encoded Tetrazine Ligation

Public Deposited

Downloadable Content

Download PDF
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/db78tk644

Descriptions

Attribute NameValues
Creator
Abstract
  • Immobilized proteins are integral components of a number of important technologies, including the principal component of many biosensors, as functional coatings in various implants, as catalysts is industrial biosynthesis, and as the building blocks of next-generation nanotechnologies. Despite their ubiquity, relatively little is known about the processes that shape immobilized protein performance at the material surface, including the immobilization process, and the subsequent structural nature of proteins at surfaces. This lack of understanding stems from, among other reasons, a lack of tools to adequately probe such processes, and to generate immobilized protein-surfaces that are conducive to study by existing technologies. This places a severe limit on our ability to study and optimize immobilized protein function, leaving us in a “trial-and-error” approach to the design and construction of protein-based biomaterials. In order to move from a “trial-and-error” approach to a principle-guided approach, new tools to study protein-surface interactions with greater precision and detail are needed. Here, the development and application of ultrafast, site-specific, and mutually orthogonal bioorthogonal labeling approaches in combination with genetic code expansion as tools to more deeply understand the nature of protein immobilization and its impact on immobilized protein structure and function is detailed. To do so, a class of noncanonical amino acids was site-specifically encoded into proteins and their rapid reactivity with strained cycloalkenes via the inverse electron-demand Diels-Alder reaction is leveraged to enable the immobilization of such proteins in defined orientations at predictable loads. On the basis of these findings, it is hypothesized that the rapid immobilization of proteins using such an approach should be able to more effectively outcompete alternative immobilization pathways, such as denaturation and aggregation which compromise the homogeneity and net order of the resulting protein surface, by reducing the time and concentration of protein during the immobilization process. In order to test this, a new genetic code expansion tool is developed that enables the simultaneous and site-specific encoding of a tetrazine-containing, and an azide-containing noncanonical amino acid into proteins. This tool is shown to enable the effective dual labeling of proteins through the mutually orthogonal bioorthogonal inverse electron demand Diels-Alder reaction, and the strain-promoted Azide-Alkyne coupling reactions and is applied for the dual labeling of proteins inside living cells. Using this tool in combination with cysteine-maleimide labeling enabled the site-specific labeling of a human carbonic anhydrase enzyme at three distinct sites, allowing not only the immobilization of this protein using vastly different immobilization reactions, but the monitoring of this process using single-molecule total internal reflection fluorescence microscopy via site-specifically installed Forster resonance energy transfer probes. Through the use of this tool, it was directly observed that faster immobilization reactions confer greater activity and structure retention, and that this is mediated through a marked reduction in the Euclidian search distance that the protein undergoes before immobilization—effectively minimizing the changes of experiencing immobilization. These results illustrate the power and potential of genetic code expansion to provide solutions that facilitate a more detailed study of protein-surface interactions. Using such an approach may serve as an important steppingstone in expanding our understanding and optimization of immobilized protein technologies.
License
Resource Type
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Rights Statement
Publisher
Peer Reviewed
Language

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details BednarRileyM2021.pdf 2021-06-18 Public Download