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Date Permissions Signed
Date of Award
Department or Program Affiliation
Master of Science (MS)
Smirnov, Sergey L.
Antos, John M.
Young, Jeff C. (Jeffery C.)
A reported 33% of eukaryotic proteins are predicted to contain intrinsically disordered regions (IDRs) over 30 residues in length. IDRs are regions of protein which natively exist in an unfolded conformation. Due to their highly dynamic nature, many common methods of inquiry such as crystallography and NMR can be thwarted. As a result, valuable analysis such as probing function, dynamics and binding interfaces are unable to be performed. To minimize these problems, researchers typically study shorter IDRs. We probed fragments of plant protein villin 4 (VLN4) for covalent modifications to explore its regulation and degradation. Disordered regions of proteins have greater solvent accessibility compared to their folded counterparts. This results in various post translational modifications, a common method of protein regulation. The protein VLN4 is vital to the development of roots and understanding its regulation could greatly benefit agriculture. Thus, VLN4 was examined for degradation, ubiquitination, and phosphorylation.
Complimentarily, overcoming challenges of IDR research can be done by targeting the IDR with covalent modification via sortase-mediated ligation (SML). In NMR, large IDRs create substantial spectral overlap in the 1H dimension for backbone amide protons. By introducing isotopically labeled residues with SML, this overlap can be greatly reduced and allow for characterization of longer IDRs. Through the use of authentic IDRs with extreme pentapeptide residue properties on N- and C- terminal flanking regions of LPXTG motifs, ligation efficiencies were characterized, followed by identification and optimization of problematic sortase ligation sequences.
Intrinsically, Disordered, Proteins
Western Washington University
Subject – LCSH
Plant proteins--Analysis; Proteins--Chemical modification; Prokaryotes--Analysis; Solvation
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McCaffery, Derek, "Covalent Modifications as Targets and Means for Research in Intrinsically Disordered Proteins" (2022). WWU Graduate School Collection. 1120.