[22] Time-resolved synchrotron X-ray footprinting and its application to RNA folding

Ralston, Corie Y.; Sclavi, Bianca; Sullivan, Michael; Deras, Michael L.; Woodson, Sarah A.; Chance, Mark R.; Brenowitz, Michael

doi:10.1016/s0076-6879(00)17024-7

Cited by 85 publications

(110 citation statements)

References 22 publications

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“…2. Because of the constant concentration of hydroxyl radicals in the sample during exposure (12,(21)(22)(23), the modification reactions followed pseudo first-order kinetics; kinetic fits are shown as solid or dashed lines in Fig. 2.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

“…The concentrations of AVP, pVIc, and DNA were measured as described previously (19,20). Samples were exposed to white light x-rays from the National Synchrotron Light Source beamline X-28C at the Brookhaven National Laboratory (Upton, NY) as described and then frozen (12,(21)(22)(23).…”

Section: Methodsmentioning

confidence: 99%

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

Gupta

Mangel

McGrath

et al. 2004

Molecular & Cellular Proteomics

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“…X-ray-dependent footprinting experiments were performed as described (21,22) at beamline X28C at the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY). Folding reactions were in 10 mM Na-cacodylate (pH 7.5), 1 mM EDTA plus 15 mM MgCl 2 (final) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme

Rangan

Masquida

Westhof

et al. 2003

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

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135

166

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Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from Azoarcus pre-tRNA ile . Base pairing of the ribozyme core requires 10-fold less Mg 2؉ than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37°C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the Azoarcus ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4 -P6 and P3-P9 domains, that may explain the unusual stability of the Azoarcus ribozyme and the cooperativity of folding.RNA modeling ͉ RNA structure ͉ metal ions ͉ hydroxyl radical footprinting T he assembly of RNA into functional structures underlies many steps in gene expression and regulation. Recent work has outlined the Mg 2ϩ -dependent folding pathways of large ribozymes (1, 2). Experimental and theoretical results suggest that the initial association of divalent cations induces collapse of the extended RNA chain into more compact structures that favor formation of tertiary interactions (3-8). The structure of the compact intermediates and the extent to which these interactions lead to the native conformation are not yet characterized.On the one hand, individual domains of the Tetrahymena group I ribozyme and the catalytic domain of RNase P fold on a time scale (10-100 ms) similar to the initial collapse transitions of the ribozyme (6, 9, 10). On the other hand, nonspecific collapse results in an ensemble of native and non-native conformations. Many larger RNAs, such as the Tetrahymena group I and Bacillus subtilis RNase P ribozymes, fold slowly in vitro because a large fraction of the RNA population becomes trapped in misfolded intermediates (11,12). An important question is whether RNAs with a simpler 3D architecture are more likely to fold directly to the native structure, as expected from theoretical models (4). Furthermore, we want to know whether the likelihood of misfolding can be predicted from the specificity of counterion-induced collapse.We investigated the Mg 2ϩ -dependent equilibrium folding pathway and constructed a 3D model of the Azoarcus group I intron. The 205-nt group IC3 intron in the pre-tRNA ile of the Azoarcus bacterium is the smallest known self-splicing group I intron (13). It retains the conserved catalytic core common to all group I introns, but lacks the peripheral domains that stabilize folding intermediates of the larger Tetrahymena intron (14).We observed two macroscopic folding transitions in the Azoarcus ribozyme...

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“…Synchrotron X-ray Radiolysis-Radiolysis experiments were performed at beamline X28C of the National Synchrotron Light Source of the Brookhaven National Laboratory (Long Island, NY) (1,27,28). An electronic shutter equipped with a platinum/iridium alloy aperture (Vincent Associates, Rochester, NY) was used to control the millisecond exposure times.…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of Gelsolin Using Synchrotron Protein Footprinting

Kiselar

Janmey

Almo

et al. 2003

Molecular & Cellular Proteomics

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101

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Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca 2؉ -activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca 2؉ -dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca 2؉ -insensitive and Ca 2؉ -dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation. Molecular & Cellular Proteomics 2:1120 -1132, 2003.Hydroxyl radicals, generated through the radiolysis of water using synchrotron radiation, can efficiently oxidize aromatic and sulfur-containing amino acids on the surface of proteins in direct relation to their reactivity and their solvent accessibility (1, 2). In particular, the solvent-accessible side chains of cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, and leucine provide convenient probes for synchrotron footprinting experiments (3). These residues cover around 30% of the sequence of a typical protein and thus provide multiple probes that offer significant coverage of the entire macromolecule (prowl.rockefeller.edu/). The radiolytic footprinting approach is illustrated in Fig. 1. Following digestion of irradiated protein solutions with proteases, the products are separated by reverse-phase liquid chromatography and analyzed by electrospray ionization mass spectrometry. The amounts of modified and unmodified peptides are individually quantitated for a specific exposure time, and the rate of oxidation of peptides containing the suscep...

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[22] Time-resolved synchrotron X-ray footprinting and its application to RNA folding

Cited by 85 publications

References 22 publications

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

DNA Binding Provides a Molecular Strap Activating the Adenovirus Proteinase

Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme

Structural Analysis of Gelsolin Using Synchrotron Protein Footprinting

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