X-ray Radiolysis Research Articles

Structural Allostery and Binding of the Transferrin·Receptor Complex

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

Structure and Dynamics of the Actin Filament

The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.

Synthesis of Nanostructured Metal−Organic Films: Surface X-ray Radiolysis of Silver Ions Using a Langmuir Monolayer as a Template

An application of the radiolysis method using an X-ray synchrotron beam is developed as a novel approach to the synthesis of metal-organic films with controlled shapes and thickness. We demonstrate that a Langmuir monolayer deposited onto a silver ion containing subphase, irradiated by an incident beam impinging below the critical angle for total reflection, induces the synthesis of a stable nanostructured silver-organic ultrathin film at the air-water interface. The X-ray scattering is also used to monitor in situ the structure of the silver layer during the synthesis process. The layer is observed by atomic force microscopy after its transfer onto a silicon substrate. One observes a film thickness of 4.6 nm, in good agreement with the X-ray penetration depth, about 4.5 nm. The silver structure is oriented by the initial organic film phase. This experiment demonstrates the considerable potential of this approach to produce various controlled metal-organic films with a surfactant self-assembly as a template.

Structural Reorganization of Proteins Revealed by Radiolysis and Mass Spectrometry: G-Actin Solution Structure Is Divalent Cation Dependent

The solution structures of isolated monomeric actins in their Mg(2+)-ATP and Ca(2+)-ATP bound forms and in complexes with gelsolin segment-1 have been probed using hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry analysis of 28 peptides containing 58 distinct reactive probe sites within actin were used to monitor conformational variations linked to divalent cation and gelsolin segment-1 binding. The solvent accessibilities of the probe sites, as measured by footprinting in solution for the Ca(2+)-G-actin and Mg(2+)-G-actin complexes with gelsolin segment-1, were consistent with available crystallographic data. This included a specific protection at the contact interface between the partners, as revealed by reduced reactivity of peptide 337-359 in the complex. Aside from the specific protection indicated previously, the oxidation rates for the reactive residues of the isolated Ca(2+)-G-actin were similar to those of the actin gelsolin segment-1 complexes; however, the reactivity of numerous residues in the isolated Mg(2+)-G-actin form was significantly reduced. Specifically, Mg(2+)-G-actin has a set of protected sites relative to Ca(2+)-G-actin that suggest a structural reorganization in subdomains 4 and 2 and a C-terminus more closely packed onto subdomain 1. These conformational variations for isolated Mg(2+)-G-actin provide a structural basis for its greater tendency to polymerize into filaments as compared to Ca(2+)-G-actin.

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.

Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting.

Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed.

ESR detection of OH radicals generated inside cells by X-ray and evaluation of the cytotoxicity of DMPO

An attempt was made to identify OH radicals inside cells in which the radicals were generated by X-ray radiolysis. In the experiments, HL60 cells were incubated for 1 hour in a medium containing 100mM 5,5-dimethyl-a-pyrroline-1-oxide (DMPO). Cells were subjected with 20Gy radiation exposure after which the cells were centrifuged. The centrifuged cells were packed in an ESR quartz column and the ESR spectrum was measured. The ESR spectrum of OH radicals was obtained 5 minutes after the irradiation; however, the ESR spectrum of DMPO-OH was weak and decreased rapidly with time in this cell-containing system. The results indicate that the main difficulty in applying the spin-trapping technique to oxygen-derived radicals formed in cell arise from the chemical instability of the resulting spin adducts. To improve the technique to study cellular systems, it will be necessary to increase the stability of the generated spin adduct.

Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the structure of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions using synchrotron light modify amino acid side-chains on millisecond timescales. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side-chains to the bulk solvent. The aromatic and sulphur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for a number of proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates correlate closely with a theoretical measure of the accessibility of residue side-chains to the solvent in the native protein structure. This approach can distinguish the relative accessibility of the tryptophan residue side-chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues, and phenylalanine at position 34 from phenylalanine residues at positions 3 and 38 based upon their rates of oxidation.

Characterisation and Properties of Amorphous Nonstoichiometric Ge1−xCx:H Compounds Obtained from X-Ray Radiolysis of Germane/Ethylene Mixtures

Characterisation and Properties of Amorphous Nonstoichiometric Ge1−xCx:H Compounds Obtained from X-Ray Radiolysis of Germane/Ethylene Mixtures

Characterisation and Properties of Amorphous Nonstoichiometric Ge1−xCx:H Compounds Obtained from X-Ray Radiolysis of Germane/Ethylene Mixtures

Compounds of amorphous hydrogenated nonstoichiometric germanium carbide were obtained from the radiolysis of GeH4/C2H4 mixtures containing 3−30% ethylene. The composition of the materials varied depending on the ethylene percentage in the irradiated mixture. The bonding and the structure of the compounds so obtained were investigated by infrared spectroscopy, and their optical gap values determined by the Tauc procedure. The variation of optical gap values as a function of carbon content is discussed.

ChemInform Abstract: Radiolytic Generation of Organic Radical Anions Isolated in Argon Matrix

Isolated radical anions of 1,3,5,7-cyclooctatetraene, 1,4-benzoquinone, 1,4-naphthoquinone, and tetracyanoethylene have been generated in argon matrices by X-ray radiolysis in the presence of amine hole traps and characterized by UV-vis absorption spectroscopy. Careful removal of water enhances the degree of conversion to the anion in the steady state, but even under the best of circumstances, the conversion is far from complete. Naphthalene, anthracene, and acenaphthylene were not converted to their radical anions to a detectable degree. It thus appears that only substrates with relatively high electron affinity can produce a significant amount of radical anions under these conditions. All observed radical anions were readily bleached back to the neutral parent with UV-vis light.

Radiolytic generation of organic radical anions isolated in argon matrix

Isolated radical anions of 1,3,5,7-cyclooctatetraene, 1,4-benzoquinone, 1,4-naphthoquinone, and tetracyanoethylene have been generated in argon matrices by X-ray radiolysis in the presence of amine hole traps and characterized by UV-vis absorption spectroscopy. Careful removal of water enhances the degree of conversion to the anion in the steady state, but even under the best of circumstances, the conversion is far from complete. Naphthalene, anthracene, and acenaphthylene were not converted to their radical anions to a detectable degree. It thus appears that only substrates with relatively high electron affinity can produce a significant amount of radical anions under these conditions. All observed radical anions were readily bleached back to the neutral parent with UV-vis light.

The specific heat at constant volume of liquid neon

The processes occurring in the X-irradiated C2H2/Kr and HCl/Kr systems in the temperature range of 7–30 K were studied using a combination of FTIR and EPR spectroscopy. In both cases, irradiation results in effective decomposition of isolated molecules (C2H2 or HCl) and production of trapped H atoms. The thermal decay of trapped atoms in solid krypton was attributed to “local” reactions (below 21 K) and long-range mobility activated in the temperature range of 23–27 K. Two krypton hydrides, HKrCCH and HKrCl, were synthesized from the radiation-induced hydrogen atoms. In the case of C2H2/Kr system, competitive reaction channels of H atoms at various absorbed doses were investigated in details, and HKrCCH was found to be one of the main reaction products. The X-ray radiolysis in krypton matrices was concluded to be a promising method to obtain krypton hydrides.