Synchrotron Radiolysis and Mass Spectrometry:  A New Approach to Research on the Actin Cytoskeleton

Guan, Jing Qu; Almo, Steven C.; Chance, Mark R.

doi:10.1021/ar0302235

Cited by 43 publications

(46 citation statements)

References 57 publications

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“…4, a and b), where the extent of protection (Ͼ90%) was consistent with burial of the respective probe sites in a macromolecular interface (12)(13)(14)30). However, ligand-induced reorganization of structure may also contribute to burial of these residues (15)(16)(17)31). The buried probe residues are indicated in Fig.…”

Section: Fig 4 Ribbon Diagram Representation Of the Avp-pvic Complexmentioning

confidence: 58%

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

Gupta

Mangel

McGrath

et al. 2004

Molecular & Cellular Proteomics

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Human adenovirus proteinase (AVP) requires two cofactors for maximal activity: pVIc, a peptide derived from the C terminus of adenovirus precursor protein pVI, and the viral DNA. Synchrotron protein footprinting was used to map the solvent accessible cofactor binding sites and to identify conformational changes associated with the binding of cofactors to AVP. The binding of pVIc alone or pVIc and DNA together to AVP triggered significant conformational changes adjacent to the active site cleft sandwiched between the two AVP subdomains. In addition, upon binding of DNA to AVP, it was observed that specific residues on each of the two major subdomains were significantly protected from hydroxyl radicals. Based on the locations of these protected side-chain residues and conserved aromatic and positively charged residues within AVP, a three-dimensional model of DNA binding was con-

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Section: Fig 4 Ribbon Diagram Representation Of the Avp-pvic Complexmentioning

confidence: 58%

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

Gupta

Mangel

McGrath

et al. 2004

Molecular & Cellular Proteomics

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“…The method is well understood to provide a sensitive probe of protein side chain accessibility, and thus residues predicted to be buried in the monomer structure as well as those involved in oligomeric contacts should be protected from covalent modification by hydroxyl radicals. In the experiment, proteins are exposed to reagents that covalently modify side chains and then digested with proteolytic enzymes; the fragments are examined for overall mass shifts indicating modification, and tandem MS is used to identify the specific sites of modification (41)(42)(43). Trypsin digestion of the protein followed by liquid chromatography-coupled MS yielded ϳ78% coverage of HR1-54Q amino acids, including all residues from the N terminus to Lys 665 .…”

Section: Volume 285 • Number 31 • July 30 2010mentioning

confidence: 99%

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Shi

Bohon

Han

et al. 2010

Journal of Biological Chemistry

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Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.The HIV 2 glycoprotein is initially expressed as the precursor protein gp160, which is post-translationally cleaved into two non-covalently associated proteins: gp120, the receptor-binding protein that targets the CD4 receptor, and gp41, which mediates the fusion of the viral and host cellular membranes (1, 2). The native, prefusion form of the gp120-gp41 complex is thought to be a trimer comprising three gp120 subunits and three membrane-anchored gp41 subunits and is in a metastable conformation with the heavily glycosylated gp120 shielding gp41 (3, 4). Upon binding of gp120 to CD4 and a co-receptor (CCR5 or CXCR4), gp41 undergoes a large conformational change to form the prehairpin fusion intermediate in which the N-terminal and C-terminal helices separate from each other and the fusion peptide extends toward the host membrane (1, 5). This ultimately leads to the fusion of the viral and host membranes and leaves a postfusion form of gp41 with a structure of hairpins in a trimer.Several crystal structures of HIV-1 and simian immunodeficiency virus gp41 have been reported (6 -9). All structures contain the gp41 core, which is composed of the two heptad repeat regions, the N-terminal helical heptad repeat (HR1) and the C-terminal helical heptad repeat (HR2). The largest structur...

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“…We used oxidative protein footprinting and mass spectrometry to probe the effect of ATP and WASp on the conformation of the Arp2/3 complex in solution with resolution at the level of single side chains (15)(16)(17). In protein complexes, the reactive residues buried between interfaces are protected from oxidation, making it possible to detect changes in surface accessibility.…”

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confidence: 99%

Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

Kiselar

Mahaffy

Pollard

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprinting with mass spectrometry in solution to probe the effects of nucleotide-and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches.

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Synchrotron Radiolysis and Mass Spectrometry: A New Approach to Research on the Actin Cytoskeleton

Cited by 43 publications

References 57 publications

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

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