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Argyris Politis

Bio: Argyris Politis is an academic researcher from King's College London. The author has contributed to research in topics: Membrane protein & Membrane lipids. The author has an hindex of 23, co-authored 59 publications receiving 2268 citations. Previous affiliations of Argyris Politis include University of Oxford & Mansfield University of Pennsylvania.


Papers
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Journal ArticleDOI
21 Oct 2011-Science
TL;DR: It is shown that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum and can link specific lipid and nucleotide binding with distinct regulatory roles.
Abstract: The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.

249 citations

Journal ArticleDOI
TL;DR: It is found that all four macromolecular complexes retain their native-like topologies at low energy during collisional activation, using ion mobility-mass spectrometry (IM-MS), and this has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.
Abstract: Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy Upon increasing the collision energy, two of the four complexes adopt a more compact state This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally

230 citations

Journal ArticleDOI
TL;DR: A detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus is reported, using a combination of electron microscopy, mass spectrometry, and deep sequencing that reveals a three-dimensional model that includes a helical component strikingly reminiscent of the backboneructure of the type I (Cascade) family.

192 citations

Journal ArticleDOI
TL;DR: Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.
Abstract: Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

148 citations

Journal ArticleDOI
TL;DR: Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.
Abstract: F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid 'plug' in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

131 citations


Cited by
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Journal ArticleDOI
28 Nov 2014-Science
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

4,774 citations

Journal ArticleDOI
TL;DR: An approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes is presented.
Abstract: The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

1,988 citations

Journal ArticleDOI
15 Sep 2016-Nature
TL;DR: Powerful mass-spectrometry-based technologies now provide unprecedented insights into the composition, structure, function and control of the proteome, shedding light on complex biological processes and phenotypes.
Abstract: Numerous biological processes are concurrently and coordinately active in every living cell. Each of them encompasses synthetic, catalytic and regulatory functions that are, almost always, carried out by proteins organized further into higher-order structures and networks. For decades, the structures and functions of selected proteins have been studied using biochemical and biophysical methods. However, the properties and behaviour of the proteome as an integrated system have largely remained elusive. Powerful mass-spectrometry-based technologies now provide unprecedented insights into the composition, structure, function and control of the proteome, shedding light on complex biological processes and phenotypes.

1,458 citations

Journal ArticleDOI
14 Mar 2014-Science
TL;DR: To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes and Actinomyces naeslundii and AnaCas9 were determined by x-ray crystallography and three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas 9:RNA:DNA were obtained by negative-stain single-particle electron microscopy.
Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

1,034 citations

Journal ArticleDOI
TL;DR: Ion mobility-mass spectrometry can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms.
Abstract: Ion mobility-mass spectrometry (IM-MS) is enhancing many areas of (bio)chemical analysis because it can separate ions both by their mass-to-charge ratio and differences in their cross-sectional area. IM-MS can be used for structural characterization, enhanced analysis of complex mixtures or to gain insights into conformational dynamics. Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis.

750 citations