Resolving intrinsically disordered proteins of the cancer genome with ion mobility mass spectrometry

For proteins the link between their structure and their function is a central tenet of biology. A common approach to understanding protein function is to ‘solve’ its structure and subsequently probe interactions between the protein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their entire structure fail to fold into a three-dimensional structure and yet they possess function. These so called intrinsically or inherently disordered proteins (IDP’s) or intrinsically disordered regions (IDR’s) constitute up to 40% of all expressed proteins. IDPs which have crucial roles in molecular recognition, assembly, protein modification and entropic chain activities, are often dynamic with respect to both conformation and interaction, so in the course of a protein’s ‘lifespan’ it will sample many configurations and bind to several targets. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. The solvent free environment of a mass spectrometer is ideally suited to the study of intrinsic interactions and how they contribute to structure. Ion mobility mass spectrometry is uniquely able to observe the range of structures an IDP can occupy, and also the effect of selected binding partners on altering this conformational space. This thesis details the technique of ion mobility mass spectrometry and illustrates its use in assessing the relative disorder of p53 protein. The tumour suppressor p53 is at the hub of a plethora of signalling pathways that maintain the integrity of the human genome and regulate the cell cycle. Deregulation of this protein has a great effect on carcinogenesis as mutated p53 can induce an amplified epigenetic instability of tumour cells, facilitating and accelerating the evolution of the tumour. Herein mass spectrometry provides a compelling, detailed insight into the conformational flexibility of the p53 DNA-binding domain. The plasticity of the p53 DNA-binding domain is reflected in the existence of more than one conformation, independent of any conformational changes prompted by binding. The in vacuo conformational phenotypes exhibited by common cancer-associated mutations are determined and the second-site suppressor mutation from loop L1, H115N, is probed whether it could trigger conformational changes in p53 hotspot cancer mutations. The structural basis of the binding promiscuity of p53 protein is investigated; of particular interest is the molecular interaction of the p53 N-terminus with the oncoprotein murine double minute 2, as well as with the antiapoptotic factor B-cell lymphoma-extralarge

Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5

Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM–MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM–MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI–MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H]+ to [M + 21H]+ and [M + 24H]+ to [M + 29H]+. The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm2 for the lower charges and 66 ± 6 nm2 for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53–54 nm2 following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm2. These data demonstrate that IM–MS is a valuable tool in studying proteins with different discrete conformational states

Structural studies of metal ligand complexes by ion mobility-mass spectrometry

Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1. 1 % and 0. 7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods. © 2013 Springer-Verlag Berlin Heidelberg

Using ion mobility spectrometry-mass spectrometry to decipher the conformational and assembly characteristics of the hepatitis B capsid protein.

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150-183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50-111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis

Structural studies of metal ligand complexes by ion mobility-mass spectrometry

The final publication is available at Springer via http://dx.doi.org/10.1007/s12127-013-0122-8Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1. 1 % and 0. 7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods. © 2013 Springer-Verlag Berlin Heidelberg

The N-Terminal residues 43 to 60 form the interface for dopamine mediated α-synuclein dimerisation

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson's disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43-140) and C-terminally (1-95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA: α-syn oligomers, albeit 1-95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43-140 protein, we analysed the structural characteristics of the DA:α-syn 43-140 dimer and α-syn 43-140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers

Ion mobility spectrometry-mass spectrometry (IMS-MS) of small molecules: separating and assigning structures to ions

The phenomenon of ion mobility (IM), the movement/transport of charged particles under the influence of an electric field, was first observed in the early 20th Century and harnessed later in ion mobility spectrometry (IMS). There have been rapid advances in instrumental design, experimental methods, and theory together with contributions from computational chemistry and gas-phase ion chemistry, which have diversified the range of potential applications of contemporary IMS techniques. Whilst IMS-mass spectrometry (IMS-MS) has recently been recognized for having significant research/applied industrial potential and encompasses multi-/cross-disciplinary areas of science, the applications and impact from decades of research are only now beginning to be utilized for "small molecule" species. This review focuses on the application of IMS-MS to "small molecule" species typically used in drug discovery (100-500 Da) including an assessment of the limitations and possibilities of the technique. Potential future developments in instrumental design, experimental methods, and applications are addressed. The typical application of IMS-MS in relation to small molecules has been to separate species in fairly uniform molecular classes such as mixture analysis, including metabolites. Separation of similar species has historically been challenging using IMS as the resolving power, R, has been low (3-100) and the differences in collision cross-sections that could be measured have been relatively small, so instrument and method development has often focused on increasing resolving power. However, IMS-MS has a range of other potential applications that are examined in this review where it displays unique advantages, including: determination of small molecule structure from drift time, "small molecule" separation in achiral and chiral mixtures, improvement in selectivity, identification of carbohydrate isomers, metabonomics, and for understanding the size and shape of small molecules. This review provides a broad but selective overview of current literature, concentrating on IMS-MS, not solely IMS, and small molecule applications. © 2012 Wiley Periodicals, Inc

ArdA proteins from different mobile genetic elements can bind to the EcoKI Type i DNA methyltransferase of E. coli K12

AbstractAnti-restriction and anti-modification (anti-RM) is the ability to prevent cleavage by DNA restriction–modification (RM) systems of foreign DNA entering a new bacterial host. The evolutionary consequence of anti-RM is the enhanced dissemination of mobile genetic elements. Homologues of ArdA anti-RM proteins are encoded by genes present in many mobile genetic elements such as conjugative plasmids and transposons within bacterial genomes. The ArdA proteins cause anti-RM by mimicking the DNA structure bound by Type I RM enzymes. We have investigated ArdA proteins from the genomes of Enterococcus faecalis V583, Staphylococcus aureus Mu50 and Bacteroides fragilis NCTC 9343, and compared them to the ArdA protein expressed by the conjugative transposon Tn916. We find that despite having very different structural stability and secondary structure content, they can all bind to the EcoKI methyltransferase, a core component of the EcoKI Type I RM system. This finding indicates that the less structured ArdA proteins become fully folded upon binding. The ability of ArdA from diverse mobile elements to inhibit Type I RM systems from other bacteria suggests that they are an advantage for transfer not only between closely-related bacteria but also between more distantly related bacterial species