Determining Surface Oriented SRCD Signatures of Proteins on DNA Nanostructures
Determining Surface Oriented SRCD Signatures of Proteins on DNA Nanostructures
Enzyme and protein activity is affected by their relative orientation when immobilised on surfaces. Localising proteins on a well-behaved surface makes it possible to study their orientation and by extension, their structure, which is pursued using oriented Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy.
SRCD is a powerful and sensitive tool to investigate the structure of proteins. Using the B23 beamline at Diamond Light Source and the novel CD imaging (CDi), methods for the preparation and measurement of oriented SRCD samples are developed in order to characterise them on surfaces. Proteins bound to two-dimensional (2D) DNA nanostructures are analysed in solution as well as absorbed on glass surfaces as films. The 2D DNA nanostructures are designed to form long-range arrays for consistent orientation over the entire sample area. Both the DNA on its own and functionalised with protein are studied in order to investigate if the alignment of the proteins on the DNA lattices can result in the change of the SRCD signature obtained from the proteins.
For this thesis, two DNA two-tiled systems are chosen, which self-assemble into 2D nanogrids and double-crossover (DX) arrays. As proof-of-concept proteins, Green Fluorescent Protein (GFP) and Cytochrome b562 are used which are site-specifically modified with azidophenylalanine, which allows to precisely position an azide at a defined residue for a single site DNA attachment using click chemistry.
Finally, a new approach of sample preparation is investigated as an alternative to the film formation, which is based on the immobilisation of biomolecules in a reversible manner and which relies on non-covalent fluorous-fluorous interactions. This method can overcome challenges that are faced during film formation which is particularly important for protein studies under dry conditions.
University of Southampton
Xyrafaki, Christina
6d784eca-f088-4536-ae4f-a248ad03534f
June 2021
Xyrafaki, Christina
6d784eca-f088-4536-ae4f-a248ad03534f
Stulz, Eugen
9a6c04cf-32ca-442b-9281-bbf3d23c622d
Xyrafaki, Christina
(2021)
Determining Surface Oriented SRCD Signatures of Proteins on DNA Nanostructures.
University of Southampton, Doctoral Thesis, 218pp.
Record type:
Thesis
(Doctoral)
Abstract
Enzyme and protein activity is affected by their relative orientation when immobilised on surfaces. Localising proteins on a well-behaved surface makes it possible to study their orientation and by extension, their structure, which is pursued using oriented Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy.
SRCD is a powerful and sensitive tool to investigate the structure of proteins. Using the B23 beamline at Diamond Light Source and the novel CD imaging (CDi), methods for the preparation and measurement of oriented SRCD samples are developed in order to characterise them on surfaces. Proteins bound to two-dimensional (2D) DNA nanostructures are analysed in solution as well as absorbed on glass surfaces as films. The 2D DNA nanostructures are designed to form long-range arrays for consistent orientation over the entire sample area. Both the DNA on its own and functionalised with protein are studied in order to investigate if the alignment of the proteins on the DNA lattices can result in the change of the SRCD signature obtained from the proteins.
For this thesis, two DNA two-tiled systems are chosen, which self-assemble into 2D nanogrids and double-crossover (DX) arrays. As proof-of-concept proteins, Green Fluorescent Protein (GFP) and Cytochrome b562 are used which are site-specifically modified with azidophenylalanine, which allows to precisely position an azide at a defined residue for a single site DNA attachment using click chemistry.
Finally, a new approach of sample preparation is investigated as an alternative to the film formation, which is based on the immobilisation of biomolecules in a reversible manner and which relies on non-covalent fluorous-fluorous interactions. This method can overcome challenges that are faced during film formation which is particularly important for protein studies under dry conditions.
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Published date: June 2021
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Local EPrints ID: 455568
URI: http://eprints.soton.ac.uk/id/eprint/455568
PURE UUID: 06ee6b17-1631-4044-b681-01e574cc1257
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Date deposited: 25 Mar 2022 17:43
Last modified: 17 Mar 2024 07:13
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Author:
Christina Xyrafaki
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