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Development and application of nitrogen-14 solid-state NMR for the analysis of biological molecules

Development and application of nitrogen-14 solid-state NMR for the analysis of biological molecules
Development and application of nitrogen-14 solid-state NMR for the analysis of biological molecules
Solid-state NMR has progressed significantly in recent decades into a powerful tool for the analysis of structure and dynamics in complex biological molecules such as proteins. The vast majority of solid-state NMR experiments on proteins rely on the isotopic enrichment of the spin-½ isotopes, 13C and 15N. However, there is a >99% naturally abundant, NMR active, isotope of nitrogen, 14N, which has a nuclear spin of 1. Its large quadrupole coupling generally precludes its use in biological NMR experiments, as it is challenging to manipulate and detect directly, though the quadrupolar coupling is highly sensitive to the local electrostatic environment. Recently, a class of solid-state NMR experiments have been introduced where 14N is studied in the indirect dimension of a 2D experiment using a spin-½ nucleus such as 13C or 1H for detection. These experiments greatly benefit from the increased sensitivity and resolution afforded by detecting 14N indirectly, but currently still lack the necessary sensitivity to be generally applicable to complex biomolecules such as proteins. These experiments typically rely on delays for free evolution in order to generate and reconvert coherence between spy nuclei and 14N. In this thesis, a novel variation on this class of experiment is presented whereby coherence is generated by long, moderate amplitude RF fields applied on the 14N channel. The experiment is demonstrated detected on both 13C and 1H on small organic molecules; demonstrating increased 14N transfer efficiencies over previous HMQC-style experiments. It is shown that the technique is robust with respect to 14N RF amplitude and pulse widths, meaning a variety of sites with different 14N environments can be detected simultaneously with minimal optimisation. Furthermore, it is shown that high quality correlation spectra can be acquired with 14N lineshapes that have well-defined features. This allows for a rigorous analysis of 14N lineshapes using numerical simulations to define 14N quadrupolar coupling parameters at a variety of sites. Finally, using the indirect detection method described herein, we demonstrate indirect detection of 14N in a 56 residue microcrystalline protein, GB3. To the best of our knowledge, this is the first solid-state NMR measurement of 14N made in a full-length protein. An analysis of the magnitude and distribution of quadrupolar couplings in the protein has been performed, demonstrating that the 14N quadrupolar coupling is highly sensitive to the local electrostatic environment, with large changes in quadrupolar interaction reflecting subtle differences in hydrogen bonding in different secondary structures.
Jarvis, James
59de8efd-053f-49da-80b4-8df7b74fa325
Jarvis, James
59de8efd-053f-49da-80b4-8df7b74fa325
Williamson, Philip
0b7715c6-b60e-4e95-a1b1-6afc8b9f372a

(2015) Development and application of nitrogen-14 solid-state NMR for the analysis of biological molecules. University of Southampton, Biological Sciences, Doctoral Thesis, 221pp.

Record type: Thesis (Doctoral)

Abstract

Solid-state NMR has progressed significantly in recent decades into a powerful tool for the analysis of structure and dynamics in complex biological molecules such as proteins. The vast majority of solid-state NMR experiments on proteins rely on the isotopic enrichment of the spin-½ isotopes, 13C and 15N. However, there is a >99% naturally abundant, NMR active, isotope of nitrogen, 14N, which has a nuclear spin of 1. Its large quadrupole coupling generally precludes its use in biological NMR experiments, as it is challenging to manipulate and detect directly, though the quadrupolar coupling is highly sensitive to the local electrostatic environment. Recently, a class of solid-state NMR experiments have been introduced where 14N is studied in the indirect dimension of a 2D experiment using a spin-½ nucleus such as 13C or 1H for detection. These experiments greatly benefit from the increased sensitivity and resolution afforded by detecting 14N indirectly, but currently still lack the necessary sensitivity to be generally applicable to complex biomolecules such as proteins. These experiments typically rely on delays for free evolution in order to generate and reconvert coherence between spy nuclei and 14N. In this thesis, a novel variation on this class of experiment is presented whereby coherence is generated by long, moderate amplitude RF fields applied on the 14N channel. The experiment is demonstrated detected on both 13C and 1H on small organic molecules; demonstrating increased 14N transfer efficiencies over previous HMQC-style experiments. It is shown that the technique is robust with respect to 14N RF amplitude and pulse widths, meaning a variety of sites with different 14N environments can be detected simultaneously with minimal optimisation. Furthermore, it is shown that high quality correlation spectra can be acquired with 14N lineshapes that have well-defined features. This allows for a rigorous analysis of 14N lineshapes using numerical simulations to define 14N quadrupolar coupling parameters at a variety of sites. Finally, using the indirect detection method described herein, we demonstrate indirect detection of 14N in a 56 residue microcrystalline protein, GB3. To the best of our knowledge, this is the first solid-state NMR measurement of 14N made in a full-length protein. An analysis of the magnitude and distribution of quadrupolar couplings in the protein has been performed, demonstrating that the 14N quadrupolar coupling is highly sensitive to the local electrostatic environment, with large changes in quadrupolar interaction reflecting subtle differences in hydrogen bonding in different secondary structures.

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More information

Published date: 1 October 2015
Organisations: University of Southampton, Centre for Biological Sciences

Identifiers

Local EPrints ID: 386966
URI: http://eprints.soton.ac.uk/id/eprint/386966
PURE UUID: 42a54372-de23-4907-9c95-f1127092e4ba
ORCID for Philip Williamson: ORCID iD orcid.org/0000-0002-0231-8640

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Date deposited: 17 Feb 2016 14:01
Last modified: 06 Jun 2018 12:39

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