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Reason: Under embargo until Feb 2017. After this date a copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library

Lanthanide tags for paramagnetic NMR spectroscopy

thesis
posted on 2017-01-18, 00:35 authored by Lee, Michael David
The site-specific incorporation of paramagnetic lanthanide ions into proteins produces a number of unique effects that manifest in the proteins’ NMR spectra, including paramagnetic relaxation enhancement, residual dipolar coupling and pseudo-contact shifts. Measurement of these effects yields powerful long-range distance and angular restraints that can aid structural investigations. In order to utilize these restraints in a wide range of proteins, synthetic lanthanide-binding tags(LBTs) capable of site-specific conjugation to proteins have been developed. This thesis describes the synthesis and assessment of nine new macrocyclic LBTs (including two pairs of enantiomeric tags) for paramagnetic NMR spectroscopy of biomolecules. Each cyclen-based LBT forms highly stable, polydenate lanthanide chelates, eliminating the need for the addition of free paramagnetic metal ions to protein samples. Factors such as the size, rigidity, hydrophobicity and method of conjugation were taken into consideration with each LBT design, resulting in the generation of a panel of high-performance tags suitable for a range of biological applications. All of the tags are designed for conjugation to cysteine residues via disulfide bond formation, except for one incorporating an α-bromoacetamide group, which generates a more stable thioether linkage. Two stand-out LBTs – the enantiomers C7 and C8 – feature very short linkers that restrict their mobility relative to the bound protein, resulting in large paramagnetic effects. Both tags were utilized in paramagnetic NMR-based structural studies of the folate biosynthesis enzyme, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, helping to establish important details about the conformation of the loops surrounding the active site. Due to their enantiomeric nature, the tags yielded two independent sets of restraints from a single tagging site.

Awards: Vice-Chancellor's Commendation for Doctoral Thesis in Excellence in 2016.

History

Campus location

Australia

Principal supervisor

Bim Graham

Additional supervisor 1

James Swarbrick

Year of Award

2015

Department, School or Centre

Medicinal Chemistry

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Pharmacy and Pharmaceutical Sciences

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