235 research outputs found
Biophysical studies into the structure and interactions of proteins and peptides
No full textInvestigating the structure of proteins and their interactions with other biomolecules or drug
molecules, coupled with the consideration of conformational change upon binding, is
essential to better understand their functions. Mass spectrometry (MS) is emerging as a
powerful tool to study protein and peptide structure and interactions due to the high dynamic
range, low sample consumption and high sensitivity of this technique, providing insight into
the stoichiometry, intensity and stability of interactions. The hybrid technique of ion
mobility-mass spectrometry (IM-MS) can provide insight into the conformations adopted by
protein and peptide monomers and multimers, in addition to complexes resulting from
interactions, which when coupled with molecular modelling can suggest candidate
conformations for these in vacuo species and by inference their conformations in solution
prior to ionisation and desolvation. The work presented in this thesis considers a number of
different peptide and protein systems, highlighting how the combination of MS and IM-MS
based techniques, in conjunction with other biophysical techniques such as circular
dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and isothermal
titration calorimetry (ITC) can provide insight into these dynamic systems.
First a case study into the ability of MS and IM-MS to study disorder-to-order transitions is
presented. The transcription factor c-MYC can only perform its function upon binding with
its binding partner MAX; deregulation of c-MYC is, however, implicated in a number of
human cancers. c-MYC and MAX comprise intrinsically disordered regions which form a
leucine zipper upon binding. The work presented here focuses on the leucine zipper regions
of both c-MYC and MAX, their individual conformations and changes upon binding.
Inhibiting the c-MYC:MAX interaction is a current target for drug therapy and hence the
inhibition of this interaction with a previously identified small drug-like molecule was also
examined using these techniques, to determine if such an approach may be appropriate for
investigation of future therapeutics.
Next the ability of MS-based techniques to preserve, transmit and distinguish between
multiple conformations of a metamorphic protein was examined. The chemokine
lymphotactin has been shown to exist in two distinct conformations in equilibrium in a
ligand-free state. The existence of such metamorphic proteins has called into question
whether traditional structural elucidation tools have been inadvertently biased towards
consideration of single conformations. Here, the potential of gas-phase techniques in the
study of conformationally dynamic systems is examined through the study of wild type lymphotactin and a number of constructs designed either as a minimum model of fold or to
mimic one of the distinct folds.
Interactions between chemokines and glycosaminoglycans (GAGs) are thought to be
essential for the in vivo activity of these proteins. The interactions between the distinctive
chemokine lymphotactin and a model GAG were hence probed. As with the structural
studies, additional protein constructs were considered either to represent the minimum model
of fold, one distinct fold of the metamorphic protein or designed to diminish its GAG
binding propensity. The ability of each construct to bind GAGs, the stoichiometry of the
interactions and conformations adopted by the resulting complexes in addition to aggregation
occurring upon the introduction of the GAG is considered.
Finally, the similarities, with respect to structure and function, between the chemokine
superfamily of proteins and the human β-defensin subfamily of antimicrobial peptides are
considered. The tendency of human β-defensins 2 and 3 to bind a model GAG is examined;
the stoichiometry of binding and conformations adopted and aggregation occurring here are
considered and compared with that of chemokines
Initial protein unfolding events in Ubiquitin, Cytochrome c and Myoglobin are revealed with the use of 213 nm UVPD coupled to IM-MS
No full textThe initial stages of protein unfolding may reflect the stability of the entire fold, and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation, experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M+7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions while cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles
Investigations of peptide structural stability in vacuo
No full textGas-phase analytical techniques provide very valuable tools for tackling the
structural complexity of macromolecular structures such as those encountered in
biological systems. Conformational dynamics of polypeptides and polypeptide
assemblies underlie most biological functionalities, yet great difficulties arise when
investigating such phenomena with the well-established techniques of X-ray
crystallography and NMR. In areas such as these ion mobility interfaced with mass
spectrometry (IMMS) and molecular modelling can make a significant contribution.
During an IMMS experiment analyte ions drift in a chamber filled with an inert gas;
measurement of the transport properties of analyte ions under the influence of a weak
electric field can lead to determination of the orientationally-averaged collision
cross-section of all resolved ionic species. A comparison with cross-sections
estimated for model molecular geometries can lead to structural assignments. Thus
IMMS can be used effectively to separate gas-phase ions based on their
conformation. The drift tube employed in the experiments described herein is
thermally regulated, which also enables the determination of collision cross-sections
over a range of temperatures, and can provide a view of temperature-dependent
conformational dynamics over the experimental (low microsecond) timescale.
Studies described herein employ IMMS and a gamut of other MS-based techniques,
solution spectroscopy and – importantly – molecular mechanics simulations to assess
a) conformational stability of isolated peptide ions, with a focus on small model
peptides and proteins, especially the Trp cage miniprotein; and b) structural
characteristics of oligomeric aggregates of an amyloidogenic peptide.
The results obtained serve to clarify the factors which dominate the intrinsic stability
of non-covalent structure in isolated peptides and peptide assemblies. Strong
electrostatic interactions are found to play a pivotal role in determining the
conformations of isolated proteins. Secondary structures held together by hydrogen
bonding, such as helices, are stable in the absence of solvent, however gas-phase
protein structures display loss of their hydrophobic cores. The absence of a polar
solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded
state observed in solution are routinely detected, indicating the existence of
intrinsically stable compact non-native states in globular proteins, illuminating the
nature of proteins’ ‘unfolded’ states
Investigation of protein‐ion interactions by mass spectrometry and ion mobility mass spectrometry
No full textProtein‐ion interactions play an important role in biological systems. A
considerable number of elements (estimated 25 – 30) are essential in higher
life forms such as animals and humans, where they are integral part of
enzymes involved in plethora of cellular processes. It is difficult to
overestimate the importance of thorough understanding of how protein‐ion
interplay affects living cell in order to be able to address therapeutic
challenges facing humanity. Presented to the reader’s attention is a gas‐phase
biophysical analysis of peptides’ and proteins’ interactions with biologically
relevant ions (Zn2+ and I–). This investigation provides an insight into
conformational changes of peptides and proteins triggered by ions.
Mass spectrometry and ion mobility mass spectrometry are used in this work
to probe peptide and protein affinities for a range of ions, along with
conformational changes that take place as a result of binding. Observation of
peptide and protein behaviour in the gas phase can inform the investigator
about their behaviour in solution prior to ionisation and transfer from the
former into the latter phase. Wherever relevant, the gas‐phase studies are
complemented by molecular dynamics simulations and the results are
compared to solution phase findings (spectroscopy).
Two case studies of protein‐ion interactions are presented in this thesis.
Firstly, sequence‐to‐structure relationships in proteins are considered via protein design approach using two synthetic peptide‐based systems. The
first system is a synthetic consensus zinc finger sequence (vCP1) that is
responsive to zinc: it adopts a zinc finger fold in the presence of Zn2+ by
coordinating the metal ion by two cysteines and two histidines. This peptide
has been selected as a reference for the zinc‐bound state and a simple model
to refine the characterisation method in preparation for analysis of a more
sophisticated second system – dual conformational switch. This second
system (ZiCop) is designed to adopt either of the two conformations in
response to a stimulus: zinc finger or coiled coil. The reversible switch
between the two conformational states is controlled by the binding of zinc
ion to the peptide. Interactions of both peptide systems with a number of
other divalent metal cations (Co2+, Ca2+ and Cu2+) are considered also, and the
differences in binding and switching behaviour are discussed. Secondly,
protein‐salt interactions are investigated using three proteins (lysozyme,
cytochrome c and BPTI) using variable temperature ion mobility mass
spectrometry. Ion mobility measurements were carried out on these proteins
with helium as the buffer gas at three different drift cell temperatures –
‘ambient’ (300 K), ‘cold’ (260 K) and ‘hot’ (360 K), and their conformational
preferences in response to HI binding and temperature are discussed
Active-metal template synthesis of a molecular trefoil knot
No full textTying the knot: The marriage of catalysis and coordination chemistry enables two CuI ions (red; see picture) to work in partnership for the synthesis of a molecular trefoil knot. One ion entangles an acyclic building block to create a loop in the ligand, and the other gathers the ligand's reactive end-groups, threads the loop, and catalyzes the covalent capture of the knotted architecture by an alkyne–azide “click” reaction
Rapid screening of diverse biotransformations for enzyme evolution
No full textThe lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence
Biophysical studies to elucidate structure-activity relationships in β-defensins
No full textβ-defensins are a class of mammalian defence peptides with therapeutic potential
because of their ability to kill bacteria and attract host immune cells. In order to
realise this potential, it is necessary to understand how the functions of these peptides
are related to their structures. This thesis presents biophysical analysis of β-
defensins and related peptides in conjunction with biological assays. These studies
provide new insights into the structure-activity relationships of β-defensins.
Ion mobility-mass spectrometry (IM-MS) is used throughout this thesis to probe the
tertiary structure of peptides in vacuo and, by inference, make conclusions about
their conformations in solution prior to ionisation. Where appropriate, IM-MS is
complemented by other techniques, including high performance liquid
chromatography and circular dichroism spectroscopy.
First, the importance of a C-terminal cysteine residue within the murine β-defensin
Defb14 is investigated. The functional and structural implications of chemically
modifying the cysteine residue are examined. Second, the N-terminal region of
Defb14 is modified by the substitution and deletion of amino acids. Again, the
effects on biological activity and structure are discussed.
Finally, the functional and structural overlap of β-defensins with another family of
proteins – the chemokines – is considered. The oligomerisation of β-defensins and
their interaction with glycosaminoglycans is of particular interest: structural data for
human β-defensins 2 and 3 in the absence and presence of polysaccharides are
presented
Bile Salts Enhance the Susceptibility of the Peach Allergenic Lipid Transfer Protein, Pru p 3, to in vitro gastrointestinal proteolysis
No full textSensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Prup3 resulting in an ensemble of around 100 peptides spanning the proteins sequence which were linked by disulphide bonds into structures of ~5-6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates
Understanding protein–drug interactions using ion mobility–mass spectrometry
No full textIon mobility–mass spectrometry (IM–MS) is an important addition to the analytical toolbox for the structural evaluation of proteins, and is enhancing many areas of biophysical analysis. Disease-associated proteins, including enzymes such as protein kinases, transcription factors exemplified by p53, and intrinsically disordered proteins, including those prone to aggregation, are all amenable to structural analysis by IM–MS. In this review we discuss how this powerful technique can be used to understand protein conformational dynamics and aggregation pathways, and in particular, the effect that small molecules, including clinically-relevant drugs, play in these processes. We also present examples of how IM–MS can be used as a relatively rapid screening strategy to evaluate the mechanisms and conformation-driven aspects of protein:ligand interactions
Luminescent, enantiopure, phenylatopyridine iridium-based coordination capsules
No full textThe first molecular capsule based on an [Ir(ppy)2]+ unit (ppy = 2-phenylatopyridine) has been prepared. Following the development of a method to resolve rac-[(Ir(ppy)2Cl)2] into its enantiopure forms, homochiral Ir6L4 octahedra where obtained with the tritopic 1,3,5-tricyanobenzene. Solution studies and X-ray diffraction show that these capsules encapsulate four of the six associated counteranions and that these can be exchanged for other anionic guests. Initial photophysical studies have shown that an ensemble of weakly coordinating ligands can lead to luminescence not present in comparable mononuclear systems
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