Developing methodology for segmental isotopic labeling of large disordered polypeptides
Research Mentor(s)
Smirnov, Serge L.
Description
We are set to utilize sortase enzymes to engineer segmentally labeled (15N, 13C) fragments into intrinsically disordered polypeptides for function/structure NMR studies. Intrinsically disordered protein fragments are actively researched today because of their prominence in human and other eukaryotic genomes. NMR spectroscopy is a powerful tool to probe disordered proteins but is problematic due to severe spectral overlap typical for larger-size samples. This severe spectral overlap can be reduced by utilizing segmental labeling with NMR-active isotopes. This allows for simplified spectra and provides greater resolving power for NMR spectroscopy of disordered proteins. To engineer these segmentally labelled protein samples, we plan to employ sortase enzymes; which can hydrolyze and re-ligate the polypeptide chains, providing a potentially powerful tool. For this project, the focus will be on proteins from the villin family of cytoskeleton regulators that contain both ordered domains (headpiece) and disordered regions (linkers). We aim to utilize a sortase site within a villin polypeptide where a folded domain headpiece connects with a disordered linker to hydrolyze and re-ligate isotopically labeled and unlabeled fragments into a larger construct. This allows us to analyze the disordered 15N-labeled linker while it is still attached the unlabeled adjacent Domain. The new construct will be used to probe by NMR the sensitivity of the linker to relevant ions. Preliminary data demonstrated proof of concept; cleavage of the headpiece domain from the linker via sortase was demonstrated and is supported by NMR and mass spectroscopy. The re-ligation of the cleaved headpiece to various linker constructs was successful as evident by mass spectroscopy.
Document Type
Event
Start Date
17-5-2018 9:00 AM
End Date
17-5-2018 12:00 PM
Department
Chemistry
Genre/Form
student projects, posters
Subjects – Topical (LCSH)
Proteins--Structure; Proteins--Conformation; Nuclear magnetic resonance spectroscopy
Type
Image
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this documentation for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.
Language
English
Format
application/pdf
Developing methodology for segmental isotopic labeling of large disordered polypeptides
We are set to utilize sortase enzymes to engineer segmentally labeled (15N, 13C) fragments into intrinsically disordered polypeptides for function/structure NMR studies. Intrinsically disordered protein fragments are actively researched today because of their prominence in human and other eukaryotic genomes. NMR spectroscopy is a powerful tool to probe disordered proteins but is problematic due to severe spectral overlap typical for larger-size samples. This severe spectral overlap can be reduced by utilizing segmental labeling with NMR-active isotopes. This allows for simplified spectra and provides greater resolving power for NMR spectroscopy of disordered proteins. To engineer these segmentally labelled protein samples, we plan to employ sortase enzymes; which can hydrolyze and re-ligate the polypeptide chains, providing a potentially powerful tool. For this project, the focus will be on proteins from the villin family of cytoskeleton regulators that contain both ordered domains (headpiece) and disordered regions (linkers). We aim to utilize a sortase site within a villin polypeptide where a folded domain headpiece connects with a disordered linker to hydrolyze and re-ligate isotopically labeled and unlabeled fragments into a larger construct. This allows us to analyze the disordered 15N-labeled linker while it is still attached the unlabeled adjacent Domain. The new construct will be used to probe by NMR the sensitivity of the linker to relevant ions. Preliminary data demonstrated proof of concept; cleavage of the headpiece domain from the linker via sortase was demonstrated and is supported by NMR and mass spectroscopy. The re-ligation of the cleaved headpiece to various linker constructs was successful as evident by mass spectroscopy.