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Protein Engineering for Novel Functionalities and for Biomolecular Applications to the Study of Protein Tyrosine O-Sulfation
Abstract
Protein tyrosine O-sulfation (PTS) is a common tyrosine post-translational modification (PTM) on many transmembrane and secreted proteins in eukaryotes. Essential recognition properties are conferred by PTS, facilitating protein-protein interactions involved in many biological and pathological processes. The major focus of this Ph. D. thesis is on the engineering and application of the Src Homology 2 (SH2) domain as an anti-sulfotyrosine antibody mimic. As compared to commercial anti-sulfotyrosine antibodies, engineered SH2 domain are smaller, less expensive, and exhibit greater affinity for PTS. In Chapter 1, a short review is provided of known tyrosine sulfated proteins (sulfoproteins), the biological significance of PTS and the current methods and biomolecular tools used for the study of PTS. In Chapter 2, the Src-SH2 domain was engineered for improved affinity and specificity for sulfotyrosine. Several SH2 variants were identified and further characterized. One SH2 variant, SH2-3.1, exhibited 15-fold greater affinity for sulfotyrosine than phosphotyrosine. In Chapter 3, the Src-SH2 domain was engineered for modulation of the preferred downstream sequence following modified tyrosine. Multiple SH2 libraries were constructed and selected against two sulfopeptides derived from CXCR4. In Chapter 4, a sulfoprotein enrichment method was devised using a photo-crosslinking unnatural amino acid to overcome non-specificity of previously identified anti-sulfotyrosine antibody mimic, SH2-60.1. Analysis of PTS using mass spectrometry has been difficult due to the labile nature of this modification and the presence of the isobaric PTM, phosphotyrosine. In collaboration with Dr. Maia Kelly from Prof. Eric Dodd’s Lab, positive mode mass spectrometry was used to identify stabilization and dissociation characteristics of model sulfopeptides as various metal adducted charge carrier states in Chapter 4. Furthermore, the application of ion mobility and collision cross section characteristics to discriminate between isobaric PTMs, sulfotyrosine and phosphotyrosine, was explored.In Chapter 6, a high-throughput method for the generation of orthogonal protein pairs was developed in collaboration with Dr. Wei Niu and Dr. Xi Song. Development of a suitable negative selection host facilitated the identification of orthogonal cohesin and dockerin protein pairs. Further application of this high-throughput method was attempted for the generation of orthogonal barnase and barstar protein pairs.
Subject Area
Chemistry
Recommended Citation
Lawrie, Justin, "Protein Engineering for Novel Functionalities and for Biomolecular Applications to the Study of Protein Tyrosine O-Sulfation" (2021). ETD collection for University of Nebraska-Lincoln. AAI28489785.
https://digitalcommons.unl.edu/dissertations/AAI28489785