Publications by year
In Press
Kish M, Smith V, Subramanian S, Vollmer F, Lethbridge N, Cole L, Bond NJ, Phillips JJ (In Press). 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.
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Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Hooper PJ, Mela I, Lashuel HA, Phillips JJ, et al (In Press). 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.
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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.
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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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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).
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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
© 2016 the Authors. Published by Elsevier Ltd. 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.
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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.
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State-of-the-art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Defining the next generation of analytical and biophysical techniques
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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.
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Characterization of Monoclonal Antibody Aggregates and Emerging Technologies
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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.
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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).
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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.
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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.
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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.
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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.
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