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NMR of peptides and proteins

NMR of peptides and proteins
NMR of peptides and proteins

The main focus of this thesis is the use of NMR for determining structural, electronic and magnetic properties of proteins and peptides. Several aspects of structure determination are investigated and new protocols are proposed and tested. The essential aim of these methods is to use as much correct experimental information as possible and check the quality of the structures using other methods as well as the standard ones such as Ramachandran plots.

In chapter one, some of the most relevant topics of NMR and a series of two-dimensional experiments are explained. The second half of this chapter deals with the standard methods used for determination of constraints and calculation and analysis of structures.

Chapters two and three involve the structure determination of a novel lantibiotic and an EGF/TFGα chimera respectively. These structure determinations use a program which takes upper and lower volume limits as input instead of distance limits. An alternative treatment for the case of completely overlapping peaks and degenerate methylene protons is presented. Extra constraints are introduced by analysing the structure for interproton distances under 2.5 Å for which there were no constraints. The volumes at the expected positions of such cross peaks were measured wherever possible. This work also uses fast-flipping aromatic residues and flexible prolines.

The remaining three chapters deal with work performed on haem proteins. Chapter four presents a model used to analyse 13C shifts of the haem substituents. This model can be used to obtain magnetic, structural and electronic information about the protein. 1H dipolar shifts are shown to be a useful way of determining the quality of the structures.

Chapter five uses the model presented in chapter four to analyse the temperature dependence of the haem orientation of cyanoferricytochrome c. Five tetrahaem cytochrome c3 proteins are used to investigate the influence of the axial ligands on the magnetic properties. The analysis revealed that the orientation of the axial ligands can be obtained from analysis of the 13C Fermi contact shifts of the haem substituents.

Chapter six reports the solution structure of the oxidised cytochrome c3 from Desulfovibrio gigas. A new protocol is used for the structure determination which involves the procedures described in chapters two and three plus the use of flexible haems, the correction of volumes for paramagnetic leakage, use of dipolar shifts as constraints and the use of angle constraints, determined from 13C data of the haems, to define the histidine orientations.

University of Southampton
Brennan, Lorraine
efa9251d-9605-48ae-b1b0-27129a4a9270
Brennan, Lorraine
efa9251d-9605-48ae-b1b0-27129a4a9270

Brennan, Lorraine (1998) NMR of peptides and proteins. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The main focus of this thesis is the use of NMR for determining structural, electronic and magnetic properties of proteins and peptides. Several aspects of structure determination are investigated and new protocols are proposed and tested. The essential aim of these methods is to use as much correct experimental information as possible and check the quality of the structures using other methods as well as the standard ones such as Ramachandran plots.

In chapter one, some of the most relevant topics of NMR and a series of two-dimensional experiments are explained. The second half of this chapter deals with the standard methods used for determination of constraints and calculation and analysis of structures.

Chapters two and three involve the structure determination of a novel lantibiotic and an EGF/TFGα chimera respectively. These structure determinations use a program which takes upper and lower volume limits as input instead of distance limits. An alternative treatment for the case of completely overlapping peaks and degenerate methylene protons is presented. Extra constraints are introduced by analysing the structure for interproton distances under 2.5 Å for which there were no constraints. The volumes at the expected positions of such cross peaks were measured wherever possible. This work also uses fast-flipping aromatic residues and flexible prolines.

The remaining three chapters deal with work performed on haem proteins. Chapter four presents a model used to analyse 13C shifts of the haem substituents. This model can be used to obtain magnetic, structural and electronic information about the protein. 1H dipolar shifts are shown to be a useful way of determining the quality of the structures.

Chapter five uses the model presented in chapter four to analyse the temperature dependence of the haem orientation of cyanoferricytochrome c. Five tetrahaem cytochrome c3 proteins are used to investigate the influence of the axial ligands on the magnetic properties. The analysis revealed that the orientation of the axial ligands can be obtained from analysis of the 13C Fermi contact shifts of the haem substituents.

Chapter six reports the solution structure of the oxidised cytochrome c3 from Desulfovibrio gigas. A new protocol is used for the structure determination which involves the procedures described in chapters two and three plus the use of flexible haems, the correction of volumes for paramagnetic leakage, use of dipolar shifts as constraints and the use of angle constraints, determined from 13C data of the haems, to define the histidine orientations.

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Published date: 1998

Identifiers

Local EPrints ID: 463444
URI: http://eprints.soton.ac.uk/id/eprint/463444
PURE UUID: f00762ec-a833-409c-b0a5-1eed6bff6ccf

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Date deposited: 04 Jul 2022 20:51
Last modified: 04 Jul 2022 20:51

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Contributors

Author: Lorraine Brennan

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