The majority of mass spectrometry analysis of biological systems is focussed on analysis of primary structure. This powerful ‘bottom-up’ approach has reaped vast benefits in post-genomic science, in particular in quantification and in locating post-translational modifications. This area of research has not much been influenced by any desire to understand conformation, and indeed most methodologies will destroy any residual fold, although indirect information on the protein fold could be obtained.
At the other end of the scale of protein architectures, mass spectrometry based analysis of quaternary protein structure, has shown that complex heterogenic topologies can be maintained into a solvent free environment.
The middle two levels of protein structure are somewhat less tractable. At the secondary structure level there is strong evidence for α-helix retention, and as yet no direct evidence for β- sheet retention. There is certainly evidence for preservation of tertiary fold but also for collapse and elongation. Much of our group research is focused at probing secondary and tertiary folds.
We have a number of active areas of investigation.
Intrinsically Disordered Protein Systems (e.g. Architecture of p53 and MDM2)
p53 the cellular tumour suppressor p53 known as ‘the guardian of the genome’ has been subject of a vast amount of research since its discovery and its analysis continues to be central to the fight against cancer. Upon a cellular stress, activation of p53 may trigger cell cycle arrest, apoptosis and senescence, thereby destroying the DNA-damaged cells before they can proliferate. The protein consists of 393 amino acids and contains several distinct domains: the N-terminal transactivation domain, a proline rich domain, the core DNA binding domain, the tetramerization domain and the C-terminal regulatory domain. Although the core DNA binding domain (DBD) forms a so called ‘ordered’ region of p53, it possesses many disordered loops and it is in these that most p53 inactivation mutations associated with nearly 50% of human cancer occur. Targeted studies into this core binding domain of oncogenic p53 and its interaction with DNA are essential to anti-cancer drug discovery efforts.
The murine double minute 2 (MDM2) is a multi-functional, multi-domain protein primarily characterized as an E3 ubiquitin ligase targeting the tumour suppressor p53. MDM2 is a 55 kDa protein which has N-terminal domain with a hydrophobic pocket, a disordered central acidic domain, a zinc finger, and a C-terminal RING domain which has similarity to other members of the RING E3 ligase family. Overall the full-length MDM2 protein is predicted to have a high level of disorder, particularly in the acidic domain which may contribute to the difficulties encountered in determining the structure of full-length MDM2. Being the binding partner of p53 makes MDM2 a promising target for cancer therapeutics.
Pre fibrillar oligomers from amyloid proteins
Amyloid fibrils are composed predominantly of a single protein or peptide and have a “cross-β” structure where the β-sheet fibril core is perpendicular to the fibril axis. A diverse range of diseases, known as amyloidoses, are characterised by amyloid fibril presence, most of which are incurable. Amyloid diseases are classified into neurodegenerative conditions, and non-neuropathic localised and systemic amyloidoses depending on the location of the amyloid plaques. In systemic and localised amyloidosis large quantities of fibrils build up, disrupting organ function. However for neurodegenerative conditions, studies have shown that pathogenesis can occur before the presence of mature fibrils. Aggregate plaques may be the end product of a process in which the principle disease-causing species are small, soluble oligomers that form early in the aggregation process.
The phenomenology of fibrillar growth, commonly observed via the binding of dyes with an affinity for hydrophobic beta-sheets, such as thioflavin T, allows the division of such processes in two principal classes: those comprising of a lag phase, exhibiting sigmoidal kinetics and those in which such a lag phase is absent. These qualitative aspects of the kinetics of amyloidogenesis must be interpreted in terms of the energetics of the assembly pathway. Simple decay kinetic profiles may point to a downhill polymerisation process or a nucleated growth process with very small energy barriers to the formation of a critical nucleus, followed by rapid fibrillar growth. Sigmoidal kinetics, which are typically associated with autocatalytic or multi-step processes, point to a mechanism consisting of formation of a population of critical nuclei, followed by rapid growth; it is know known that “secondary nucleation processes”, such as fibril fragmentation, influence significantly the shape of the fibril growth curve.
Although the structure of mature fibrils is well characterised, the precise details of the process by which a soluble protein or peptide converts to fibrillar form are elusive. MS is a valuable technique for investigating this phenomenon as it can separate the species present in the lag phase, identifying the many heterogenic, transiently populated oligomeric orders according to their m/z ratios revealing their relative abundance. The hybrid technique IM-MS combined with molecular modelling enhances this picture by probing the conformational landscape of the aggregating system.
Therapeutics – Stability of Protein Complexes
Despite an increasing success of ion mobility mass spectrometry (IM-MS) to study intact biomolecules, there are still some controversies regarding structure preservation in the gas phase and data calibration obtained from the commercially available travelling wave ion mobility spectrometry (TWIMS) instrument Synapt HDMS. The issue with any mobility analyzer used to investigate the conformations of biomolecules is that the measurement itself may alter the conformation of the ion. Application of high injection energies and long ion storage times can cause structure collapse or reorganisation via unfolding. Recent studies show, that the ion effective temperature within TWIMS for small proteins can exceed 500 K which will cause an ion to re-order to more elongated conformations.
Variable temperature IM-MS is used to gain more insights into the thermal stability of mutlimeric protein complexes in the gas phase. Our data is acquired on the home built linear DT-IM-MS instrument at different buffer gas (helium) temperatures ranging from 200 to 500 K.
Large protein complexes such as transthyretin (55 kDa), avidin (64 kDa), concanavalin A (102.6 kDa) or serum amyloid P component (256 kDa) have been investigated to examine their thermal stability, collapse, unfolding and complex dissociation. Preliminary data acquired at 400 K shows a slight decrease in collision cross sections (CCSs) of the complexes in comparison to CCSs determined at 300 K, suggesting a structural collapse. Moreover, the stochastic increase in CCSs usually observed with increasing charge state resulting from protein unfolding due to coulombic repulsions, is no longer observable, again supporting the complex collapse.
When exposed to temperatures above 500 K, investigated protein complexes dissociate into subunits. Capturing the ions prior to the dissociation (at 470 – 480 K) reveals a drastic increase in the CCSs associated with protein unfolding. Based on the variable temperature IM-MS data, we speculate that investigated protein complexes exposed to high temperatures collapse first prior to unfolding.
Peptides/Biophysics of Protein fold e.g. Switching Peptides, effects of anions
To be added…
Chemokines and antimicrobial peptides
Chemokines are a class of small secreted proteins which interact with G protein-coupled receptors. Chemokines can be induced during immune response for the recruitment of immune system cells to the site of an infection or during the control of cell migration for tissue maintenance and development. We are interested in studying a unique chemokine, lymphotactin, in addition to a class of chemotactic peptides known as β-defensins.
Lymphotactin is the only member of the C sub class of chemokines and is involved in the recruitment of T and NK cells. Lymphotactin induces chemotaxis and calcium mobilization through binding with the G protein-coupled receptor known as XCR1. It contains a unique extended C terminus sequence, which forms an intrinsically disordered tail. Additionally, it assumes two distinct conformations in equilibrium, with interconversion between these two folds involving a complete restructuring of the core residues.
Interestingly, each structure performs separate roles; the monomeric, conserved chemokine fold, activates the XCR1, whilst the unique dimeric conformation, is required for binding of extracellular glycosaminoglycans. The equilibrium between the two conformations can be altered through ionic strength or specific mutations.
In addition we are also involved in the study of β-defensins, which are an interesting class of chemotactic and antimicrobial peptides. Defensins are an important class of cysteine-rich cationic peptides, which function in antimicrobial defense. These antimicrobial peptides are found in many different species including plants, mammals and insects, and have been found to display broad-spectrum activity towards both Gram-positive and Gram-negative bacteria in addition to fungi and enveloped viruses. Defensins vary with respect to the number of cysteine residues present, in addition to the resulting disulfide linkages. Mammalian defensins however are composed of six cysteine residues and can be subdivided into three distinct class; α-, β- and θ-defensins, based on the connectivity of the disulfide bridges observed. Β-defensins are described as having cysteine bridges between CI-CV, CII-CIV and CIII-CIV and are primarily expressed at epithelial surfaces.
β-defensins are amphipathic peptides, containing cationic and hydrophobic regions, and are typically 30-45 amino acids in length. Their tertiary structure consists of three β-strands which are arranged in an antiparallel sheet and stabilised by the disulfide bonds. The N-terminal fragment of the peptides forms a α-helical segment which is stabilised to the β-sheet structure through the CI-CV disulfide bond. Within the second β-strand a highly conserved amino acid sequence has been found, Gly-X-CysIV, which forms a β-bulge, thought to be necessary for the correct folding.
We collaborate with the group of Dr Paul Lusby (University of Edinburgh) specialists in design of functional self-assembled supramolecular systems. Biomimetic, stimuli-responsive self-assembly is one of the most attractive of the recent developments in the field of metallosupramolecular cage systems. The scope of the possible applications of the stimuli-responsive self-assembly is vast; encapsulation/release mechanisms are required in sensing, catalysis, drug delivery and pharma. Self-assembly processes involve weak, non-covalent interactions, and these supramolecular complexes can also aggregate into higher order assembled structures, both of which can be problematic for structural analysis by X-Ray crystallography or NMR.
In addition to standard nanoESI MS we employ a range of other techniques to obtain detailed structural information about the self-assemblies. Collision Induced Dissociation (CID) experiments provide insights into the strength of the particular non-covalent interactions and fragmentation pathways. Drift Tube Ion Mobility MS complemented by computational modelling and ion mobility simulations (MOBCAL) provide powerful tools to determine three dimensional structure of the complexes.