The University of Southampton
University of Southampton Institutional Repository

Method development in biological solid-state NMR

Method development in biological solid-state NMR
Method development in biological solid-state NMR
Solid-state nuclear magnetic resonance (ssNMR) has proved to be a useful tool in the analysis of structural and dynamic properties of proteins. However, the inherent low sensitivity of NMR hinders further advancements of the field. This thesis focuses on improving the sensitivity of ssNMR, be this through an improvement in signal to noise or an effective improvement in sensitivity by enhancing the information content. The mixed rotational and rotary resonance (MIRROR) sequence1 was employed to facilitate protein backbone assignment under moderate spinning speeds. Through the band selective nature of MIRROR, bidirectional transfer of magnetisation from the CCO site to the adjacent Cα and to the Cα of the next amino acid is possible. When applied to a 3D-NCOCA experiment this may be used to double the information content, providing correlations from CCO(i-1) to both Cα(i-1) and Cα(i). The MIRROR recoupling of CCO to Cα, is inherently low-power, allowing MIRROR to be utilised in a low-power experiment. Through this, greater enhancements in sensitivity per unit time can be realised. The longitudinal relaxation time (T1) limits the sensitivity per unit time at both room and cryogenic temperatures. To develop the use of relaxation agents for cryogenic NMR experiments, the room temperature relaxation properties and dynamics of the model protein, GB3, were explored. Site-specific relaxation measurements were used to understand the relaxation of residues in the protein and gain understanding into how this relates to the dynamics of the protein. In addition, low-temperature NMR measurements were used to investigate the effect temperature has on relaxation.

The ongoing development of polarisation enhancement methods and machinery have made great progress in recent years particularly on the application towards biomolecules. However, arguably the most promising polarisation enhancement technique, dynamic nuclear polarisation (DNP) suffers from a variety of problems. Namely, line broadening effects as a result of the low-temperatures required and through doping with paramagnetic agents. Furthermore, the current method of sample preparation for DNP via the use of doping with exogenous radicals is not viable for all samples. Endogenous radicals for the DNP of large biomolecules may offer several advantages to their exogenous counterparts, including but not limited to, a greater understanding of quenching effects and polarisation transfer. This thesis explores the possibility of creating pseudo-biradicals bound covalently to a protein surface to elicit enhancements through the cross effect DNP mechanism.

In summary, we have developed a range of methods that enhance the information content and sensitivity, which will provide new approaches for researchers investigating proteins using ssNMR.
University of Southampton
Jolly, Michael Matthew
c927d250-88b0-4675-b8da-237cb0c0f0b1
Jolly, Michael Matthew
c927d250-88b0-4675-b8da-237cb0c0f0b1
Levitt, Malcolm
bcc5a80a-e5c5-4e0e-9a9a-249d036747c3

Jolly, Michael Matthew (2017) Method development in biological solid-state NMR. University of Southampton, Doctoral Thesis, 267pp.

Record type: Thesis (Doctoral)

Abstract

Solid-state nuclear magnetic resonance (ssNMR) has proved to be a useful tool in the analysis of structural and dynamic properties of proteins. However, the inherent low sensitivity of NMR hinders further advancements of the field. This thesis focuses on improving the sensitivity of ssNMR, be this through an improvement in signal to noise or an effective improvement in sensitivity by enhancing the information content. The mixed rotational and rotary resonance (MIRROR) sequence1 was employed to facilitate protein backbone assignment under moderate spinning speeds. Through the band selective nature of MIRROR, bidirectional transfer of magnetisation from the CCO site to the adjacent Cα and to the Cα of the next amino acid is possible. When applied to a 3D-NCOCA experiment this may be used to double the information content, providing correlations from CCO(i-1) to both Cα(i-1) and Cα(i). The MIRROR recoupling of CCO to Cα, is inherently low-power, allowing MIRROR to be utilised in a low-power experiment. Through this, greater enhancements in sensitivity per unit time can be realised. The longitudinal relaxation time (T1) limits the sensitivity per unit time at both room and cryogenic temperatures. To develop the use of relaxation agents for cryogenic NMR experiments, the room temperature relaxation properties and dynamics of the model protein, GB3, were explored. Site-specific relaxation measurements were used to understand the relaxation of residues in the protein and gain understanding into how this relates to the dynamics of the protein. In addition, low-temperature NMR measurements were used to investigate the effect temperature has on relaxation.

The ongoing development of polarisation enhancement methods and machinery have made great progress in recent years particularly on the application towards biomolecules. However, arguably the most promising polarisation enhancement technique, dynamic nuclear polarisation (DNP) suffers from a variety of problems. Namely, line broadening effects as a result of the low-temperatures required and through doping with paramagnetic agents. Furthermore, the current method of sample preparation for DNP via the use of doping with exogenous radicals is not viable for all samples. Endogenous radicals for the DNP of large biomolecules may offer several advantages to their exogenous counterparts, including but not limited to, a greater understanding of quenching effects and polarisation transfer. This thesis explores the possibility of creating pseudo-biradicals bound covalently to a protein surface to elicit enhancements through the cross effect DNP mechanism.

In summary, we have developed a range of methods that enhance the information content and sensitivity, which will provide new approaches for researchers investigating proteins using ssNMR.

Text
Final Version - Version of Record
Available under License University of Southampton Thesis Licence.
Download (22MB)

More information

Published date: December 2017

Identifiers

Local EPrints ID: 422129
URI: http://eprints.soton.ac.uk/id/eprint/422129
PURE UUID: 9f3f0342-6bd3-4b67-ac3c-779c0d77df80
ORCID for Malcolm Levitt: ORCID iD orcid.org/0000-0001-9878-1180

Catalogue record

Date deposited: 17 Jul 2018 16:30
Last modified: 14 Mar 2019 01:47

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×