Publications by year
In Press
Phillips J (In Press). Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions.
Journal of Visualized ExperimentsAbstract:
Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions
Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are. abundant. in. Lewy. bodies. and. neurites. which. are. the. hallmarks. of. Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the. soluble. monomer. form. of. the. protein. Elucidation. of. the. molecular. mechanisms of. aSyn. biology. and. pathophysiology. requires. structurally. highly. resolved. methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make. monomeric. aSyn. intractable. to. many. structural. biology. techniques. Here. the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry. (HDX-MS). on. the. millisecond. timescale. for. the. study. of. proteins. with low. thermodynamic. stability. and. weak. protection. factors. such. as. aSyn. At. the millisecond timescale, HDX-MS data contain information on the solvent accessibility and. hydrogen-bonded. structure. of. aSyn. which. are. lost. at. longer. labeling. times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics. and. thermodynamics. intra-. and. inter-molecular. interactions. and. thestructural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.
Abstract.
2023
Kish M, Subramanian S, Smith V, Lethbridge N, Cole L, Vollmer F, Bond NJ, Phillips JJ (2023). Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250′ and 280s Loops. Biochemistry, 62(8), 1360-1368.
Kish M, Ivory DP, Phillips JJ (2023). Allosteric activation and inhibition of glycogen phosphorylase share common transient structural features.
Abstract:
Allosteric activation and inhibition of glycogen phosphorylase share common transient structural features
ABSTRACTIt remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme, glycogen phosphorylase is one of the most studied proteins and is a clinical target of much interest to treat type II diabetes and metastatic cancers. However, a lack of understanding of its complex regulation, mediated by dynamic structural changes, hinder its exploitation as a drug target. Here, we precisely locate dynamic structural changes upon allosteric activation and inhibition of glycogen phosphorylase, by developing a time-resolved non-equilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach. We resolved obligate transient changes in localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect at a common structurally dynamic motif. This approach has broad application to determine the structural kinetic mechanisms by which proteins are regulated.
Abstract.
Kish M, Smith V, Lethbridge N, Cole L, Bond NJ, Phillips JJ (2023). Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution. Analytical Chemistry, 95(11), 5000-5008.
Seetaloo N (2023). Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease.
Abstract:
Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease
Lewy bodies – the hallmarks of Parkinson’s disease – are majorly constituted of aggregates of the presynaptic protein alpha-synuclein. The molecular mechanism of alpha-synuclein aggregation through which it changes dramatically from a soluble disordered monomer to insoluble structured fibrils remains unknown. As an intrinsically disordered protein, alpha-synuclein does not have a specific three-dimensional structure, but rather behaves mostly as a meta-stable ensemble of highly dynamic conformers, and as such undergoes rapid kinetics, making it almost impossible to measure its conformational changes with most techniques. Millisecond amide hydrogen exchange can provide valuable insights on the dynamic behaviour of proteins, especially at flexible regions. Thus, the work in this thesis reports on the development of methods and tools for hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and the application of these for the study of aSyn under physiological conditions. In the first part of this thesis, high resolution on the alpha-synuclein monomer was achieved over two dimensions: time and space. Using a novel in-house rapid- mixing quench-flow instrument, hydrogen/deuterium-exchange mass spectrometry data on alpha-synuclein on the millisecond timescale was attained. Furthermore, using a ‘soft’ gas-phase mass spectrometry fragmentation technique called Electron Transfer Dissociation, structural resolution in the protein increased. The second part of this work focuses on the development of a software, HDfleX, in an effort to primarily automate the HDX-MS workflow and allow the merging of HDX-MS data at different levels: bottom-up, middle-down and top-down. The rest of the thesis uses the tools and methods developed earlier on to explore the effects of different solution conditions (cellular compartments and salt cations) on the monomeric conformations of aSyn, and how these correlate to the different stages of aggregation and the ensuing fibril polymorphs. Altogether, the achievements in this work will allow us to better understand the plasticity of the alpha-synuclein monomer as it cycles through different local environments.
Abstract.
2022
Guo Z, Parakra RD, Xiong Y, Johnston WA, Walden P, Edwardraja S, Moradi SV, Ungerer JPJ, Ai HW, Phillips JJ, et al (2022). Engineering and exploiting synthetic allostery of NanoLuc luciferase.
Nature Communications,
13(1).
Abstract:
Engineering and exploiting synthetic allostery of NanoLuc luciferase
Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein’s active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini.
Abstract.
Seetaloo N, Zacharopoulou M, Stephens AD, Kaminski Schierle GS, Phillips JJ (2022). Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions.
Abstract:
Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions
ABSTRACTIn Parkinson’s disease and other synucleinopathies, the intrinsically disordered, presynaptic protein alpha-synuclein misfolds and aggregates. We hypothesise that the exposure of alpha-synuclein to different cellular environments, with different chemical compositions, pH and binding partners, alters its biological and pathological function by inducing changes in molecular conformation. Our custom instrumentation and software enable measurement of the amide hydrogen exchange rates of wild-type alpha-synuclein at amino acid resolution under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal compartments of cells. We characterised the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Our findings reveal that the C-terminal residues of alpha-synuclein are driving its nucleation and thus its aggregation. Furthermore, the entire NAC region and specific other residues strongly promoted elongation of fibrils. This provides new detail on our current understanding of the relationship between the local chemical environment and monomeric conformations of alpha-synuclein.
Abstract.
Orozco CT, Bersellini M, Irving LM, Howard WW, Hargreaves D, Devine PWA, Siouve E, Browne GJ, Bond NJ, Phillips JJ, et al (2022). Mechanistic insights into the rational design of masked antibodies. mAbs, 14(1).
Seetaloo N, Zacharopoulou M, Stephens AD, Kaminski Schierle GS, Phillips JJ (2022). Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein. Analytical Chemistry, 94(48), 16711-16719.
Seetaloo N, Phillips JJ (2022). Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions.
Journal of Visualized Experiments,
2022(184).
Abstract:
Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions
Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra-and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.
Abstract.
2021
Seetaloo N, Kish M, Phillips JJ (2021). HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data.
Abstract:
HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data
ABSTRACTHydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined – they can only be analyzed separately. Thus, we developed HDfleX – a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.
Abstract.
Seetaloo N, Kish M, Phillips JJ (2021). HDfleX: Software for the flexible structural resolution of hydrogen-deuterium exchange mass spectrometry data.
Abstract:
HDfleX: Software for the flexible structural resolution of hydrogen-deuterium exchange mass spectrometry data
A standalone application for the post-processing of hydrogen-deuterium exchange mass spectrometry data, including curve fitting, merging of bottom-up and middle-down data and robust statistical significance analyses, amongst others. The scientific basis and application of HDfleX has been described thoroughly in the following publication:
Seetaloo, N. Kish, M. and Phillips, J. J. (2021) ‘HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data’, bioRxiv, p. 2021.12.09.471740. doi: 10.1101/2021.12.09.471740.
Abstract.
Orozco CT, Edgeworth MJ, Devine PWA, Hines AR, Cornwell O, Thompson C, Wang X, Phillips JJ, Ravn P, Jackson SE, et al (2021). Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation. Bioconjugate Chemistry, 32(8), 1834-1844.
Groves K, Ashcroft AE, Cryar A, Sula A, Wallace BA, Stocks BB, Burns C, Cooper-Shepherd D, De Lorenzi E, Rodriguez E, et al (2021). Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.
Anal Chem,
93(26), 9041-9048.
Abstract:
Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.
Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.
Abstract.
Author URL.
2020
Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Woodhams PJ, Mela I, Lashuel HA, Phillips JJ, et al (2020). Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity.
Nature Communications,
11(1).
Abstract:
Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn’s aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
Abstract.
2019
Kish M, Smith V, Subramanian S, Vollmer F, Lethbridge N, Cole L, Bond NJ, Phillips JJ (2019). Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution.
Abstract:
Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution
AbstractGlycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. The sensitivity of these measurements discerned that the 250s’ loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site.Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability which provides direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop.FootnotesAuthor contributions: M.K. V.S. S.S. N.L. F.V. N.B. L.C. and J.J.P. designed research; M.K. V.S. S.S. L.C. and J.J.P. performed research; M.K. V.S. S.S. L.C. and J.J.P. analyzed data; and M.K. and J.J.P. wrote the manuscript.
Abstract.
Zacharopoulou M, Stephens A, Mela I, Moons R, Sobott F, Phillips J, Schierle GK (2019). Disruption of monomeric alpha-synuclein long-range interactions alters its misfolding propensity.
Author URL.
Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Hooper PJ, Mela I, Lashuel HA, Phillips JJ, et al (2019). Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity.
Abstract:
Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity
AbstractAs an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyn’s aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.
Abstract.
2018
Lautenschläger J, Stephens AD, Fusco G, Ströhl F, Curry N, Zacharopoulou M, Michel CH, Laine R, Nespovitaya N, Fantham M, et al (2018). C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.
Nat Commun,
9(1).
Abstract:
C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
Abstract.
Author URL.
Stephens AD, Nespovitaya N, Zacharopoulou M, Kaminski CF, Phillips JJ, Kaminski Schierle GS (2018). Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing.
Anal Chem,
90(11), 6975-6983.
Abstract:
Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing.
Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson's and Alzheimer's disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.
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Author URL.
2017
Phillips JJ, Buchanan A, Andrews J, Chodorge M, Sridharan S, Mitchell L, Burmeister N, Kippen AD, Vaughan TJ, Higazi DR, et al (2017). Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues. Analytical Chemistry, 89(4), 2361-2368.
2016
Dobson CL, Devine PWA, Phillips JJ, Higazi DR, Lloyd C, Popovic B, Arnold J, Buchanan A, Lewis A, Goodman J, et al (2016). Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.
Scientific Reports,
6(1).
Abstract:
Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo
AbstractUncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.
Abstract.
Millership C, Phillips JJ, Main ERG (2016). Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.
Journal of Molecular Biology,
428(9), 1804-1817.
Abstract:
Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway
Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.
Abstract.
Borisov OV (2016).
State-of-the-art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Defining the next generation of analytical and biophysical techniques., ACS Symposium.
Abstract:
State-of-the-art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Defining the next generation of analytical and biophysical techniques
Abstract.
2015
Remmele RL, Bee JS, Phillips JJ, Mo WD, Higazi DR, Zhang J, Lindo V, Kippen AD (2015). Characterization of Monoclonal Antibody Aggregates and Emerging Technologies.
Abstract:
Characterization of Monoclonal Antibody Aggregates and Emerging Technologies
Abstract.
Edgeworth MJ, Phillips JJ, Lowe DC, Kippen AD, Higazi DR, Scrivens JH (2015). Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.
Angew Chem Int Ed Engl,
54(50), 15156-15159.
Abstract:
Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.
Immunoglobulinâ€
G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.
Abstract.
Author URL.
Klein T, Vajpai N, Phillips JJ, Davies G, Holdgate GA, Phillips C, Tucker JA, Norman RA, Scott AD, Higazi DR, et al (2015). Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase.
Nature Communications,
6(1).
Abstract:
Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase
AbstractProtein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp–Phe–Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called ‘DFG flip’. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a ‘DFG-out’ state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and ‘molecular brake’ regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.
Abstract.
2014
Tavakoli-Keshe R, Phillips JJ, Turner R, Bracewell DG (2014). Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.
J Pharm Sci,
103(2), 437-444.
Abstract:
Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.
Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid-liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (Tm 1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.
Abstract.
Author URL.
2013
Main ERG, Phillips JJ, Millership C (2013). Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.
Biochem Soc Trans,
41(5), 1152-1158.
Abstract:
Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.
There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.
Abstract.
Author URL.
2012
Phillips JJ, Millership C, Main ERG (2012). Fibrous nanostructures from the self-assembly of designed repeat protein modules.
Angew Chem Int Ed Engl,
51(52), 13132-13135.
Abstract:
Fibrous nanostructures from the self-assembly of designed repeat protein modules.
Single-protein-chain superhelical filaments are obtained from monomeric repeat proteins by controlling the chemistry and solvent exposure at their terminal interfaces. The assembly was achieved in aqueous solution, at neutral pH value, and at room temperature. The building block was a recombinantly engineered designed tetratricopeptide repeat protein. Directed head-to-tail self-assembly was driven by genetically encoded orthogonal native chemical ligation.
Abstract.
Author URL.
Phillips JJ, Javadi Y, Millership C, Main ERG (2012). Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.
Protein Sci,
21(3), 327-338.
Abstract:
Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.
Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.
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2007
Phillips JJ, Yao Z-P, Zhang W, McLaughlin S, Laue ED, Robinson CV, Jackson SE (2007). Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs.
J Mol Biol,
372(5), 1189-1203.
Abstract:
Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs.
The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.
Abstract.
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