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2. Novel routes to defined post translational modifications using non-canonical amino acids

Author

Worthy, Harley Luke

Subjects
572
Abstract

Proteins are inherently limited by the properties of their constituent amino acids and attempt to overcome this by using post translational modifications (PTMs). PTMs are highly specific and can effectively modulate protein function faster than simple up or down regulation of protein production. However, PTMs often require a suite of other proteins to regulate and perform the modification to ensure accuracy, which can be hard to engineer into synthetic proteins. By introducing new chemistry into proteins via noncanonical amino acids (ncAAs) we can expand the range of new non-native PTMs that we can explore. Non-native PTMs (nnPTMs), have the potential to be both bioorthogonal and easily transferable between proteins. This thesis examines the effects of engineering nnPTMs into superfolder Green Fluorescent Protein (sfGFP) to study the effects on fluorescence of: 1) modification with small molecules (Chapter 3), 2) Creation of covalent protein dimers (Chapter4), 3) Interfacing proteins to carbon nanomaterials (Chapter 5), and 4) Look at the effects of engineering cooperativity using ncAAs (Chapter6). Most of this work focused on the ncAA, p-azido-L-phenylalanine (azF) as it has several properties that would be desirable for use in proteins such as photo reactivity and selective reactivity with alkynes. Moreover, as azF can be incorporated into any target protein in a range of hosts, it is an ideal starting point to engineer nnPTMs that are easily transferable. Throughout this thesis the importance of intricate hydrogen bonding networks and water channels, to the function of a protein, is made apparent through a range of in silico, structural and biophysical techniques. In silico modelling is used throughout to predict; the effects of nnPTMs on sfGFP structure (Chapter 3 and Chapter 6), dimer interfaces in Chapter 4, and show functional linking between sfGFP and carbon nanotubes in Chapter 5.

Published
2018

3. Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus.

Author

De Rose, Simone A., Isupov, Michail N., Worthy, Harley L., Stracke, Christina, Harmer, Nicholas J., Siebers, Bettina, and Littlechild, Jennifer A.

Subjects
HYDROXYL group, ACETOLACTATE synthase, AMINO acids, GLUTAMINE synthetase, ENZYMES, METHANOGENS
Abstract

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Å resolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications. [ABSTRACT FROM AUTHOR]

Published
2023
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4. Glycosylation increases active site rigidity leading to improved enzyme stability and turnover

Author

Ramakrishnan, Krithika, primary, Johnson, Rachel L., additional, Winter, Samuel D., additional, Worthy, Harley L., additional, Thomas, Christopher, additional, Humer, Diana C., additional, Spadiut, Oliver, additional, Hindson, Sarah H., additional, Wells, Stephen, additional, Barratt, Andrew H., additional, Menzies, Georgina E., additional, Pudney, Christopher R., additional, and Jones, D. Dafydd, additional

Published
2023
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9. enzyme structure and dynamics figures from Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Author

Noby, Nehad, Auhim, Husam Sabah, Winter, Samuel, Worthy, Harley L., Embaby, Amira M., Saeed, Hesham, Hussein, Ahmed, Pudney, Christopher R., Rizkallah, Pierre J., Wells, Stephen A., and Jones, D. Dafydd

Abstract

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical ��/�� hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Published
2021
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10. The Crystal Structure of Bacillus cereus HblL1

Author

Worthy, Harley L., primary, Williamson, Lainey J., additional, Auhim, Husam Sabah, additional, Leppla, Stephen H., additional, Sastalla, Inka, additional, Jones, D. Dafydd, additional, Rizkallah, Pierre J., additional, and Berry, Colin, additional

Published
2021
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11. Author Correction:Positive functional synergy of structurally integrated artificial protein dimers assembled by Click chemistry (Communications Chemistry, (2019), 2, 1, (83), 10.1038/s42004-019-0185-5)

Author

Worthy, Harley L., Auhim, Husam Sabah, Jamieson, W. David, Pope, Jacob R., Wall, Aaron, Batchelor, Robert, Johnson, Rachel L., Watkins, Daniel W., Rizkallah, Pierre, Castell, Oliver K., and Jones, D. Dafydd

Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Published
2019

12. Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer

Author

Pope, Jacob R., primary, Johnson, Rachel L., additional, Jamieson, W. David, additional, Worthy, Harley L., additional, Kailasam, Senthilkumar, additional, Ahmed, Rochelle D., additional, Taban, Ismail, additional, Auhim, Husam Sabah, additional, Watkins, Daniel W., additional, Rizkallah, Pierre J., additional, Castell, Oliver K., additional, and Jones, D. Dafydd, additional

Published
2020
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13. Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc00944a Click here for additional data file

Author

Hartley, Andrew M., Worthy, Harley L., Reddington, Samuel C., Rizkallah, Pierre J., and Jones, D. Dafydd

Subjects
Chemistry, fungi
Abstract

Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation., Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-l-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and original protein. Crystal structures of both the click modified and photochemically converted forms show that functional switching is due to local conformational changes that optimise the interaction networks surrounding the chromophore. Crystal structure and mass spectrometry studies of the irradiated protein suggest that the phenyl azide converts to a dehydroazepine and/or an azepinone. Thus, protein embedded phenyl azides can be used beyond simple photocrosslinkers and passive conjugation handles, and mimic many natural post-translational modifications: modulation though changes in interaction networks.

Published
2016

14. Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer.

Author

Pope, Jacob R., Johnson, Rachel L., Jamieson, W. David, Worthy, Harley L., Kailasam, Senthilkumar, Ahmed, Rochelle D., Taban, Ismail, Auhim, Husam Sabah, Watkins, Daniel W., Rizkallah, Pierre J., Castell, Oliver K., and Jones, D. Dafydd

Subjects
FLUORESCENT proteins, FLUORESCENCE resonance energy transfer, ENERGY transfer, FLUORESCENCE
Abstract

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild‐type FPs are fused to a naturally occurring protein–protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction. [ABSTRACT FROM AUTHOR]

Published
2021
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15. Novel routes to defined post translational modifications using non-canonical amino acids.

Author

Worthy, Harley Luke and Worthy, Harley Luke

Abstract

Proteins are inherently limited by the properties of their constituent amino acids and attempt to overcome this by using post translational modifications (PTMs). PTMs are highly specific and can effectively modulate protein function faster than simple up or down regulation of protein production. However, PTMs often require a suite of other proteins to regulate and perform the modification to ensure accuracy, which can be hard to engineer into synthetic proteins. By introducing new chemistry into proteins via noncanonical amino acids (ncAAs) we can expand the range of new non-native PTMs that we can explore. Non-native PTMs (nnPTMs), have the potential to be both bioorthogonal and easily transferable between proteins. This thesis examines the effects of engineering nnPTMs into superfolder Green Fluorescent Protein (sfGFP) to study the effects on fluorescence of: 1) modification with small molecules (Chapter 3), 2) Creation of covalent protein dimers (Chapter4), 3) Interfacing proteins to carbon nanomaterials (Chapter 5), and 4) Look at the effects of engineering cooperativity using ncAAs (Chapter6). Most of this work focused on the ncAA, p-azido-L-phenylalanine (azF) as it has several properties that would be desirable for use in proteins such as photo reactivity and selective reactivity with alkynes. Moreover, as azF can be incorporated into any target protein in a range of hosts, it is an ideal starting point to engineer nnPTMs that are easily transferable. Throughout this thesis the importance of intricate hydrogen bonding networks and water channels, to the function of a protein, is made apparent through a range of in silico, structural and biophysical techniques. In silico modelling is used throughout to predict; the effects of nnPTMs on sfGFP structure (Chapter 3 and Chapter 6), dimer interfaces in Chapter 4, and show functional linking between sfGFP and carbon nanotubes in Chapter 5.

16. Novel routes to defined post translational modifications using non-canonical amino acids.

Author

Worthy, Harley Luke and Worthy, Harley Luke

Abstract

Proteins are inherently limited by the properties of their constituent amino acids and attempt to overcome this by using post translational modifications (PTMs). PTMs are highly specific and can effectively modulate protein function faster than simple up or down regulation of protein production. However, PTMs often require a suite of other proteins to regulate and perform the modification to ensure accuracy, which can be hard to engineer into synthetic proteins. By introducing new chemistry into proteins via noncanonical amino acids (ncAAs) we can expand the range of new non-native PTMs that we can explore. Non-native PTMs (nnPTMs), have the potential to be both bioorthogonal and easily transferable between proteins. This thesis examines the effects of engineering nnPTMs into superfolder Green Fluorescent Protein (sfGFP) to study the effects on fluorescence of: 1) modification with small molecules (Chapter 3), 2) Creation of covalent protein dimers (Chapter4), 3) Interfacing proteins to carbon nanomaterials (Chapter 5), and 4) Look at the effects of engineering cooperativity using ncAAs (Chapter6). Most of this work focused on the ncAA, p-azido-L-phenylalanine (azF) as it has several properties that would be desirable for use in proteins such as photo reactivity and selective reactivity with alkynes. Moreover, as azF can be incorporated into any target protein in a range of hosts, it is an ideal starting point to engineer nnPTMs that are easily transferable. Throughout this thesis the importance of intricate hydrogen bonding networks and water channels, to the function of a protein, is made apparent through a range of in silico, structural and biophysical techniques. In silico modelling is used throughout to predict; the effects of nnPTMs on sfGFP structure (Chapter 3 and Chapter 6), dimer interfaces in Chapter 4, and show functional linking between sfGFP and carbon nanotubes in Chapter 5.

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