Adaptation of Chemical Biology Approaches for Dissecting Disordered Protein Dynamics

IDP;Intrinsically Disordered Proteins;Protein Dynamics;Chemistry;Science;Chemical Biology

Johnson, OletaNikolovska-Coleska, Zaneta ;

University of Michigan

关键词: IDP;    Intrinsically Disordered Proteins;    Protein Dynamics;    Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/147481/otjohn_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Proteins containing intrinsic disorder often undergo dynamic conformational change in response to ligand binding or post-translational modification. Chemical dissection of these dynamic protein perturbations presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein (IDP) that both folds into a short alpha-helix upon binding to its protein ligand, eIF4E, and a four-stranded beta-sheet upon hyperphosphorylation. Post-translational modifications (PTMs) are an important mode of regulation for IDPs and intrinsically disordered regions (IDRs); however, the enzymatic generation of uniformly modified proteins in vitro remains challenging. Studying PTMs in IDPs is further complicated by their instability and markedly dynamic nature. Chemical methods of site-specific PTM incorporation have been developed in attempt to circumvent this problem. Here, we evaluate a chemical mutagenesis-based approach for generating pCys as a phosphomimetic in the 4E-BPs, a family of proteins that acts to repress cap-dependent translation, and whose function is regulated by a hierarchy of phosphorylation events. Using NMR and CD spectroscopy, we have characterized pCys in two unique contexts within 4E-BPs: induction and destabilization of secondary structure. Understanding the applicability of pCys in these unique contexts is important for expanding its use to answer biological questions in complex protein systems.Additionally, biophysical methods for analysis of binding-induced structural changes are low throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the alpha-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.