Reductive Alkylation of Proteins Towards Structural and Biological Applications
type: CreativeWork
description: Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies, typically requiring the incorporation of stable magnetic isotopes to improve sensitivity and assign resonances. Degenerate levels of 13C-incorporation have been the biggest obstacle for mass spectrometry-assisted assignment of 13C-dimethylamine resonances in nuclear magnetic resonance spectroscopy (NMR). Reductive 13C-methylation is an alternative labeling method for proteins not amenable to bacterial host overexpression. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. The research presented in the first part of this dissertation explores several methods used to break the degeneracy in 13C-labeling of lysozyme. To overcome the degeneracy in labeling lysozyme with the reductive methylation reaction, we investigated two methods: 1) reductive methylation in the presence of 18-crown-6-ether (18C6) and 2) reductive methylation using multiple reducing agents. To assign the - and -dimethylamine resonances of the N-terminal lysine residue of lysozyme, a non-destructive Edman degradation method was explored. The second part of this research discusses an alternative assignment method based on mass spectrometry to aid in the assignment of the NMR signals from reductively 13C-methylated proteins. Because assignment is increasingly difficult when lysine is the N-terminal residue of the protein, one method is described to identify the NMR resonance of the 13C-methyls associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme Lys1 are identified in 1H-13C heteronuclear single-quantum correlation spectra. Protein chemical modification is a well-established field that continues to impact leading research today including glycomimetics and cross-linking of proteins. Cu…
abstract: Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies, typically requiring the incorporation of stable magnetic isotopes to improve sensitivity and assign resonances. Degenerate levels of 13C-incorporation have been the biggest obstacle for mass spectrometry-assisted assignment of 13C-dimethylamine resonances in nuclear magnetic resonance spectroscopy (NMR). Reductive 13C-methylation is an alternative labeling method for proteins not amenable to bacterial host overexpression. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. The research presented in the first part of this dissertation explores several methods used to break the degeneracy in 13C-labeling of lysozyme. To overcome the degeneracy in labeling lysozyme with the reductive methylation reaction, we investigated two methods: 1) reductive methylation in the presence of 18-crown-6-ether (18C6) and 2) reductive methylation using multiple reducing agents. To assign the - and -dimethylamine resonances of the N-terminal lysine residue of lysozyme, a non-destructive Edman degradation method was explored. The second part of this research discusses an alternative assignment method based on mass spectrometry to aid in the assignment of the NMR signals from reductively 13C-methylated proteins. Because assignment is increasingly difficult when lysine is the N-terminal residue of the protein, one method is described to identify the NMR resonance of the 13C-methyls associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme Lys1 are identified in 1H-13C heteronuclear single-quantum correlation spectra. Protein chemical modification is a well-established field that continues to impact leading research today including glycomimetics and cross-linking of proteins. Cu…
author: Roberson, Kevin Jerome
keywords: Chemical modification, dimethyllysine, isotope labeling, dimethylamine borane, reductive methylation
date published: 2014-01-01T08:00:00Z
date modified: 2026-07-24T02:59:15Z
provider: Lousiana State University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Chemistry

PropertyValue
property ID: DOI
value: 10.31390/gradschool_dissertations.1267