542 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
Exploring gas-phase protein conformations by ion mobility-mass spectrometry
No full textAnalysis and characterisation of biomolecules using mass spectrometry has advanced over the past decade due to improvements in instrument design and capability; relevant use of complementary techniques; and available experimental and in silico data for comparison with cutting-edge research. This thesis presents ion mobility data, collected on an in-house modified QToF mass spectrometer (the MoQTOF), for a number of protein systems.
Two haemoproteins, cytochrome c and haemoglobin, have been characterised and rotationally-averaged collision cross-sections for a number of multimeric species are presented. Intact multiply-charged multimers of the form [xCyt c + nH]z+ where x = 1 (monomer), x = 2 (dimer) and x = 3 (trimer) for cytochrome c have been elucidated and for species with x ≥ 2, reported for the first time. Fragment ions possibly attributed to a novel fragmentation mechanism, native electron capture dissociation, are reported with a brief discussion into their possible production from the dissociation of the gas-phase dimer species.
Haemoglobin monomer globin subunits, dimers and intact tetramer have been successfully transferred to the gas phase, and their cross-sections elucidated. Comparisons with in silico computational data have been made and a discussion of the biologically-active tetramer association/dissociation technique is presented.
Three further proteins have been studied and their gas-phase collision cross-sections calculated. Two regions of the large Factor H (fH) complement glycoprotein, fH 10-15 and fH 19-20, have been characterised for the first time by ion mobility-mass spectrometry. Much work using nuclear magnetic resonance spectroscopy has previously been achieved to produce structural information of these protein regions, however further biophysical characterisation using mass spectrometry may aid in greater understanding of the interactions these two specific regions have with other biomolecules.
The DNA-binding core domain of the tumour suppressor p53 has been characterised and cross-sections produced in the presence and absence of the zinc metal ion that may control the domain’s biological activity. Within this core domain, p53 inactivation mutations have been shown to occur in up to 50% of human cancers, therefore the potential exists to further cancer-fighting activity through research on this region. Anterior Gradient-2 (AGR2) protein facilitates downregulation of p53 in an as yet unclear mechanism. Recent work using peptide aptamers has demonstrated that this downregulation can be disrupted and levels of p53 restored. Collision cross-sections for six peptide aptamers have been calculated, as well as cross-sections for multimers of AGR2 protein. A complex between one aptamer with the protein has also been elucidated.
Use of the commercially available Synapt HDMS ion mobility-mass spectrometer at Waters MS Technologies Centre (Manchester, UK) allowed data to be collected for both Factor H protein regions and for the DNA-binding core domain of p53. Data are compared in the appropriate chapters with data collected using the MoQTOF
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
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
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
Angle-resolved femtosecond photoelectron spectroscopy of fullerenes
No full textAn experimental apparatus has been constructed to investigate ionisation mechanisms of complex molecules and nanoparticles after femtosecond and/or picosecond laser excitation. The photoproducts are detected by time-of-flight mass spectrometry and velocity-map imaging (VMI) photoelectron spectroscopy. Test measurements on C60 and Xe have successfully reproduced previously published work indicating that the setup is working in a satisfactory manner. New detailed investigations of mass spectra and angle resolved photoelectron spectra (PES) have been carried out as a function of laser intensity, wavelength and pulse duration for C60 and C70, providing new insights into the electronic structure and ionisation mechanisms of these molecules.
For 400 nm, 130 fs laser excitation, an isotropic contribution from thermally emitted electrons is found. A series of peaks are seen superimposed on the thermal background with binding energies in agreement with the recently discovered superatom molecular orbitals (SAMOs) of C60 [Feng et.al. Science 320 (2008) p. 359]. Furthermore, the angular dependence of the peak in the PES corresponding to the s-SAMO is in agreement with this assignment. To confirm the assignment of the other observed peaks it is concluded that the measured photoelectron angular distributions (PADs) need to be compared to calculated angular distributions. Measurements have also been made with the same wavelength but with a pulse duration of about 5 ps. Mass spectra, PES and PADs for these measurements show that the main ionisation mechanism for these laser conditions is delayed (thermionic) ionisation.
For 800 nm, 130 and 180 fs laser excitation, thermally emitted electrons are observed. In contrast to the 400 nm measurements, the PADs show an asymmetry with higher apparent temperatures along the laser polarisation direction. Measurements were also made for longer pulse durations (1.0 – 3.8 ps). For pulse durations above 1 ps the asymmetry is gradually reduced while the delayed ionisation component in the mass spectrum increases with increasing pulse duration. The asymmetry is compared to calculations made assuming a field-assisted thermal electron emission. Similarly to the 400 nm experiments, a series of peaks are seen superimposed on the thermal background. PADs are presented for these peaks. PADs for peaks with the same binding energy as peaks seen in the 400 nm experiments follow the same trend.
Isotropic PADs after ns laser excitation are also presented confirming delayed ionisation for these pulse durations
Proteomic investigation of the MDM2 interactome and linear motif interactions
No full textThe oncoprotein MDM2 has an integral role in cancer development via multiple signalling pathways. Two proteomic mass spectrometry screens, label-free with spectral counting quantitation and 8-plex iTRAQ were used to identify proteins up or downregulated over time by the MDM2 targeting drug Nutlin. A subset of previously identified MDM2 binding partners were identified as altered after Nutlin treatment, along with proteins which have not as yet been linked to MDM2 or p53. Proteins altered two hours after Nutlin treatment were screened for sequence similarity to an MDM2 binding consensus motif based on the BOX-I region of p53. Peptides corresponding to this motif were validated for MDM2 binding, and the mode of binding investigated using competition ELISA and thermal denaturation assays. Known MDM2 ligands such as Nutlin were shown to have a range of effects on the binding of these newly identified MDM2 peptides, which may be attributed to allosteric regulation of MDM2. The effects of Nutlin on two full length proteins identified by the MS screens, CypB and NPM, were confirmed in vivo. In vitro binding of MDM2 to CypB and PK, which contain BOX-I like motifs, was also demonstrated validating proteomic mass spectrometry screens as a method to identify new protein-protein interactions. To further investigate the potential of linear motifs to modulate protein-protein interactions, a peptide aptamer targeting the protein AGR2 was tested for effect on AGR2 and p53 in a cancer cell line
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
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