Neil J. Oldham

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Organization: University of Nottingham , England

Department: School of Chemistry

Title: (PhD)

Chemicals
References

Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein

Co-reporter:Lucio Manzi;Dr. Andrew S. Barrow;Dr. Jonathan T. S. Hopper;Renata Kaminska; Colin Kleanthous; Carol V. Robinson; John E. Moses; Neil J. Oldham

Angewandte Chemie International Edition 2017 Volume 56(Issue 47) pp:14873-14877

Publication Date(Web):2017/11/20

DOI:10.1002/anie.201708254

AbstractMapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study a carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane-spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein–protein interactions between the trimer subunits remained unlabelled, thus revealing their location.

Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein

Co-reporter:Lucio Manzi;Dr. Andrew S. Barrow;Dr. Jonathan T. S. Hopper;Renata Kaminska; Colin Kleanthous; Carol V. Robinson; John E. Moses; Neil J. Oldham

Angewandte Chemie 2017 Volume 129(Issue 47) pp:15069-15073

Publication Date(Web):2017/11/20

DOI:10.1002/ange.201708254

Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins

Co-reporter:Matthew Jenner, Jose P. Afonso, Christoph Kohlhaas, Petra Karbaum, Sarah Frank, Jörn Piel and Neil J. Oldham

Chemical Communications 2016 vol. 52(Issue 30) pp:5262-5265

Publication Date(Web):15 Mar 2016

DOI:10.1039/C6CC01453D

Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

Relative Binding Affinities of Integrin Antagonists by Equilibrium Dialysis and Liquid Chromatography–Mass Spectrometry

Co-reporter:William J. Tipping, Nkazimulo Tshuma, James Adams, Harvey T. Haywood, James E. Rowedder, M. Jonathan Fray, Thomas McInally, Simon J. F. Macdonald, and Neil J. Oldham

ACS Medicinal Chemistry Letters 2015 Volume 6(Issue 2) pp:221

Publication Date(Web):December 22, 2014

DOI:10.1021/ml500395v

The integrin αvβ6 is a potential target for treatment of idiopathic pulmonary fibrosis (IPF). Equilibrium dialysis (ED) was investigated for its ability to report ligand binding in an αvβ6 inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against αvβ6, and the fraction bound (fb) and percentage saturation determined by liquid chromatography–mass spectrometry (LC-MS) analysis. Quantitation of the inhibitor in the two chambers of the ED cartridge revealed an uneven distribution in the presence of αvβ6, corresponding to near saturation binding to the protein (93 ± 3%), while the control (without integrin) showed an equal partitioning of the inhibitor on either side of the dialysis membrane. A competitive ED assay with a 12 component mixture of antagonists was conducted, and the results compared with an established cell adhesion assay for quantifying αvβ6 inhibition of individual antagonists. Compounds clustered into three groupings: those with pIC50 values between ca. 5.0 and 5.5, which possessed ED fb values indistinguishable from the controls, those with pIC50s of 6.5 ± 0.2, which exhibited detectable integrin binding (fb 13–25%) in the ED assay, and a single compound of pIC50 7.2 possessing an fb value of 38%. A good correlation between ED-derived fb and pIC50 was observed despite the two assays utilizing quite different outputs. These results demonstrate that ED with LC-MS detection shows promise as a rapid αvβ6 integrin antagonist screening assay for mixtures of putative ligands.Keywords: equilibrium dialysis; Idiopathic pulmonary fibrosis; integrin; liquid chromatography−mass spectrometry; αvβ6

Acyl-Chain Elongation Drives Ketosynthase Substrate Selectivity in trans-Acyltransferase Polyketide Synthases

Co-reporter:Matthew Jenner;Dr. José Pedro Afonso;Hannah R. Bailey;Sarah Frank;Dr. Annette Kampa;Dr. Jörn Piel;Dr. Neil J. Oldham

Angewandte Chemie International Edition 2015 Volume 54( Issue 6) pp:1817-1821

Publication Date(Web):

DOI:10.1002/anie.201410219

Abstract

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl-ACP to yield an extended β-ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans-acyltransferase (trans-AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.

Acyl-Chain Elongation Drives Ketosynthase Substrate Selectivity in trans-Acyltransferase Polyketide Synthases

Co-reporter:Matthew Jenner;Dr. José Pedro Afonso;Hannah R. Bailey;Sarah Frank;Dr. Annette Kampa;Dr. Jörn Piel;Dr. Neil J. Oldham

Angewandte Chemie 2015 Volume 127( Issue 6) pp:1837-1841

Publication Date(Web):

DOI:10.1002/ange.201410219

Abstract

Amino acid-accepting ketosynthase domain from a trans-AT polyketide synthase exhibits high selectivity for predicted intermediate

Co-reporter:Christoph Kohlhaas, Matthew Jenner, Annette Kampa, Geoff S. Briggs, José P. Afonso, Jörn Piel and Neil J. Oldham

Chemical Science 2013 vol. 4(Issue 8) pp:3212-3217

Publication Date(Web):31 May 2013

DOI:10.1039/C3SC50540E

The trans-acyltransferase (AT) polyketide synthases are a recently recognised group of bacterial enzymes that generate complex polyketides. A prerequisite for re-engineering these poorly studied systems is knowledge about the substrate specificity of their components. In this work, KS domain 1 from the bacillaene polyketide synthase has been shown to possess high specificity towards 2-amidoacetyl intermediates, which are derived from incorporation of alpha amino acids into the polyketide chain. N-Acetylcysteamine (SNAC) analogues of full-length substrates were synthesised and incubated with the KS1 domain. The natural glycine-derived acyl–SNAC was found to acylate KS1 with highest efficiency, as evidenced by mass spectrometry (MS). An alanine variant was also incorporated, but its valine equivalent was not, which indicated limited tolerance of substitution at the α-position. Substrate analogues without an amine or amide nitrogen substituted on the 2-position were not accepted by KS1 at the standard assay concentration of 0.5 mM. Moreover, removal of Asn-206 from the active site of KS1 by site-directed mutagenesis reduced kcat/Km by a factor of approx. 2. This residue is conserved in most known 2-amidoacetyl-accepting KS domains from trans-AT PKSs and we postulate an important interaction between Asn-206 and the amide nitrogen of the substrate.

The effects of cation adduction upon the conformation of three-helix bundle protein domains

Co-reporter:Kleitos Sokratous;Robert Layfield

International Journal for Ion Mobility Spectrometry 2013 Volume 16( Issue 1) pp:19-27

Publication Date(Web):2013/03/01

DOI:10.1007/s12127-012-0114-0

The ubiquitin-binding, three-helix bundle domains of the proteins ubiquilin 1 (UQ1) and hHR23A both exhibited remarkably high, but discrete, ammonium ion adduction when electrosprayed from aqueous ammonium acetate. The degree of adduction was highly charge state dependent with, unusually, the lowest charge states (+3 for UQ1 and +4 for hHR23A) showing almost no adducts and the highest charge states (+5 for UQ1 and +6 for hHR23A) exhibiting adduction with two ammonium cations as the most abundant form. As the charge state of protein ions produced by electrospray ionisation (ESI) is related to solvent-accessible surface area we inferred that the ammonium-carrying ions were of a more open conformation than their protonated counterparts. This was confirmed by ESI-travelling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS), which showed that, although the purely protonated ions were compact, their equivalents bearing one or two ammonium adducts exhibited populations of significantly larger collisional cross section (CCS). We postulate that complexation with the ammonium cation may disrupt a key salt bridge(s) in the compact structure. A similar effect is observed with mono-sodium ion adduction, but this is diminished with each additional sodium ion in the complex to produce more compact structures.

Substrate Specificity in Ketosynthase Domains from trans-AT Polyketide Synthases

Co-reporter:Matthew Jenner;Sarah Frank;Annette Kampa;Christoph Kohlhaas;Petra Pöplau;Dr. Geoff S. Briggs;Dr. Jörn Piel;Dr. Neil J. Oldham

Angewandte Chemie International Edition 2013 Volume 52( Issue 4) pp:1143-1147

Publication Date(Web):

DOI:10.1002/anie.201207690

Probing Affinity and Ubiquitin Linkage Selectivity of Ubiquitin-Binding Domains Using Mass Spectrometry

Co-reporter:Kleitos Sokratous ; Lucy V. Roach ; Debora Channing ; Joanna Strachan ; Jed Long ; Mark S. Searle ; Robert Layfield

Journal of the American Chemical Society 2012 Volume 134(Issue 14) pp:6416-6424

Publication Date(Web):March 19, 2012

DOI:10.1021/ja300749d

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2–200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.

Charge state and adduct reduction in electrospray ionization–mass spectrometry using solvent vapor exposure

Co-reporter:Jonathan T.S. Hopper, Kleitos Sokratous, Neil J. Oldham

Analytical Biochemistry 2012 Volume 421(Issue 2) pp:788-790

Publication Date(Web):15 February 2012

DOI:10.1016/j.ab.2011.10.034

The benefits of lowering protein ion charge states in electrospray ionization (ESI) have attracted recent interest. We describe a simple approach to decrease protein charge states by exposure of electrospray droplets to neutral solvent vapor such as acetonitrile. The technique allows detection of weak noncovalent complexes, provides preferred charge states for tandem mass spectrometry (MS/MS) dissociation of protein complexes, and has the added benefit of reducing common adducts, such as alkali metals, without the addition of solution additives or the requirement for a secondary spray.

Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization

Co-reporter:Jonathan T. S. Hopper;Andrew Rawlings

Journal of The American Society for Mass Spectrometry 2012 Volume 23( Issue 10) pp:1757-1767

Publication Date(Web):2012 October

DOI:10.1007/s13361-012-0430-y

It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra- and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional activation of wild-type and mutant-FKBP•FK506 ions, generated by ESI, demonstrated that removal of native protein–ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex. Mutation of Arg42 to Ala42, or Tyr26 to Phe26 also resulted in lower energy dissociation of the FKBP·FK506 complex. Although these residues do not form direct H-bonds to FK506, they interact with Asp37, ensuring its correct orientation to associate with the ligand. Comparison with solution-based affinity measurements of these mutants has been discussed, including the stabilization afforded by ordered water molecules. Ion mobility spectrometry (IMS) has been employed to provide gas-phase structural information on the unfolding of the complexes. The [M + 6H]6+ complexes of the wild-type and mutants have been shown to resist unfolding and retain compact conformations. However, removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H]7+ complex when raised to dissociation-level energies. Overall, destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.

Alkali Metal Cation-Induced Destabilization of Gas-Phase Protein–Ligand Complexes: Consequences and Prevention

Co-reporter:Jonathan T. S. Hopper and Neil J. Oldham

Analytical Chemistry 2011 Volume 83(Issue 19) pp:7472

Publication Date(Web):August 24, 2011

DOI:10.1021/ac201686f

Electrospray ionization, now a well established technique for studying noncovalent protein–ligand interactions, is prone to production of alkali metal adducts. Here it is shown that this adduction significantly destabilizes the interactions between two model proteins and their ligands and that destabilization correlates with cation size. For both the [FKBP·FK506] and [lysozyme·NAGn] systems, dissociation of the metalated complex occurs at markedly lower collision energies than their purely protonated equivalents. Dependency upon size of the metal+ demonstrates the importance of electrostatic charge density during the dissociation process. Differences in the gas phase basicities (GBapp) of the multiply charged protein ions and proton and sodium affinities of the ligands explain the observed charge partitioning during dissociation of the complexes. Ion mobility-mass spectrometry measurements demonstrate that metal cation adduction does not induce a significant increase in unfolding of the polypeptides, indicating that this is not the principal mechanism responsible for destabilization. Destabilizing effects can be largely reduced by exposing the electrospray to solvent (e.g., acetonitrile) vapor, a method that acts to reduce the amount of adduct formation as well as decrease the charge states of the resulting ions. This approach leads to more accurate determination of apparent KDs in the presence of trace alkali metals.

Detection of a Protein Conformational Equilibrium by Electrospray Ionisation-Ion Mobility-Mass Spectrometry

Co-reporter:Matthew Jenner;Dr. Jacqueline Ellis;Dr. Wei-Cheng Huang;Dr. Emma LloydRaven;Dr. Gordon C. K. Roberts;Dr. Neil J. Oldham

Angewandte Chemie International Edition 2011 Volume 50( Issue 36) pp:8291-8294

Publication Date(Web):

DOI:10.1002/anie.201101077

Detection of a Protein Conformational Equilibrium by Electrospray Ionisation-Ion Mobility-Mass Spectrometry

Co-reporter:Matthew Jenner;Dr. Jacqueline Ellis;Dr. Wei-Cheng Huang;Dr. Emma LloydRaven;Dr. Gordon C. K. Roberts;Dr. Neil J. Oldham

Angewandte Chemie 2011 Volume 123( Issue 36) pp:8441-8444

Publication Date(Web):

DOI:10.1002/ange.201101077

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Co-reporter:Jonathan T. S. Hopper

Journal of The American Society for Mass Spectrometry 2009 Volume 20( Issue 10) pp:1851-1858

Publication Date(Web):2009 October

DOI:10.1016/j.jasms.2009.06.010

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.

Cyclisation of Lys48-linked diubiquitin in vitro and in vivo

Co-reporter:Kleitos Sokratous, Joanna Strachan, Lucy V. Roach, Robert Layfield, Neil J. Oldham

FEBS Letters (30 November 2012) Volume 586(Issue 23) pp:4144-4147

Publication Date(Web):30 November 2012

DOI:10.1016/j.febslet.2012.10.011

Ubiquitin (Ub) is able to form polymeric isopeptide-linked chains through condensation of any of its seven lysine (Lys) residues with the C-terminus of an adjacent Ub monomer. Electrospray ionisation mass spectrometry (ESI-MS) of commercial in vitro-generated Lys48-linked di-Ub (Lys48-Ub2) revealed a major population of cyclised dimer. The absence of a free C-terminus in this population was confirmed by an inability to bind the zinc finger ubiquitin-binding domain (ZnF-UBP) of USP5/isopeptidase-T. Endogenous Ub2 purified from skeletal muscle and cultured mammalian cells was found to contain cyclic Lys48-Ub2, demonstrating that cyclisation of poly-Ub can also occur in vivo.Highlights► Commercial Lys48-linked diubiquitin contains cyclic diubiquitin. ► Cyclic Lys48-linked diubiquitin is unable to bind to the ZnF-UBP of isopeptidase-T. ► Endogenous cyclic Lys48-linked diubiquitin is present in mammalian cells.

Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins

Co-reporter:Matthew Jenner, Jose P. Afonso, Christoph Kohlhaas, Petra Karbaum, Sarah Frank, Jörn Piel and Neil J. Oldham

Chemical Communications 2016 - vol. 52(Issue 30) pp:NaN5265-5265

Publication Date(Web):2016/03/15

DOI:10.1039/C6CC01453D

Amino acid-accepting ketosynthase domain from a trans-AT polyketide synthase exhibits high selectivity for predicted intermediate

Co-reporter:Christoph Kohlhaas, Matthew Jenner, Annette Kampa, Geoff S. Briggs, José P. Afonso, Jörn Piel and Neil J. Oldham

Chemical Science (2010-Present) 2013 - vol. 4(Issue 8) pp:NaN3217-3217

Publication Date(Web):2013/05/31

DOI:10.1039/C3SC50540E