Download or read book Development of In-situ Protein Engineering Technologies for the Study of Modified Proteins written by Michael R. Haugbro and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Deciphering the roles that protein post-translational modifications, protein-protein interactions, and protein dynamics play in modulating protein structure and function requires the ability to introduce amino acid modifications, cross-linking moieties, biophysical probes, and other synthetic chemical species into proteins. Current technologies for the semi-synthesis of modified proteins are optimized for in vitro reactions and are not easily transferable to more biologically relevant systems such as live cell culture. Analogous methodologies that work in situ rely on genetic manipulations that often result in low resolution data and a high level of off-target effects. Early attempts to address this disconnect have employed protein ligase systems such as Sortase and split inteins, but as of yet have not become robust enough for wide-spread use. In this thesis we expand upon the existing technologies and introduce new methods for the precise chemical manipulation of proteins in cell culture using split inteins. We applied in-nucleo protein trans-splicing to determine the in situ interactome of histone PTMs, combining chemical precision and native protein environment in an unprecedented manner unattainable by any other approach. The full results of the proteomics experiments enabled by this technique can be found in Appendix 2, available with the digital version of this thesis. We also expanded the in situ PTS toolbox by developing a multiplexed method for histone labeling in nuclei and in live cells. We demonstrated this capability by labeling two histones with different color fluorophores in a completely orthogonal manner. Finally, we utilized an atypical split intein known as VidaL to achieve the first semi-synthesis of a modified protein in cells, generating histones bearing multiple PTMs and an affinity handle in the chromatin of live cells. We added to this platform by combining our semi-synthesis capabilities with control of the subcellular localization of our target protein, in a single reaction. We envision that these new methods described in this thesis will equip protein chemists and cellular biologists alike with new capabilities for the study of protein structure and function.