Satoko Akashi

Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and frit-FAB LC/MS

Incorrectness of the amino acid sequence of bovine serum albumin (BSA) was suggested from the observed molecular weight of BSA obtained by electrospray ionization mass spectrometry (ESIMS). Lack of a tyrosine residue in the position of 156th was found rapidly, by the combination of frit-fast atom bombardment mass spectrometry/liquid chromatography (Frit-FAB LC/MS), automated Edman degradation and tandem mass spectrometry (MS). Then it turned out that BSA is composed of 583 amino acid residues, and that its average molecular weight is not 66267.1, and it is corrected to 66430.3. Moreover the amino acid sequence of the positions of 94th and 95th was corrected to -QE- by using automated Edman degradation method.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca2+ binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca2+ binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

Crystal Structure of Human REV7 in Complex with a Human REV3 Fragment and Structural Implication of the Interaction between DNA Polymerase ζ and REV1

DNA polymerase ζ (Polζ) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polζ consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847–1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polζ to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.

Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling

Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins–NPM1, NPM2 and NPM3–expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm–nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

A self-assembled protein nanotube with high aspect ratio.

Production of a self-assembled protein nanotube achieved through engineering of the 11mer ring protein trp RNA-binding attenuation protein is described. The produced mutant protein is able to stack in solution to produce an extremely narrow, uniform nanotube apparently stabilized by a mixture of disulfide bonds and hydrophobic interactions. Assembly is reversible and the length of tube can potentially be controlled. Large quantities of hollow tubes 8.5 nm in overall diameter with lengths varying from 7 nm to over 1 microm are produced. The structure is analyzed using transmission electron microscopy, atomic force microscopy, mass spectrometry, and single-particle analysis and it is found that component rings stack in a head-to-head fashion. The internal diameter of the tube is 2.5 nm, and the amino acid residues lining the central cavity can be mutated, raising the possibility that the tube can be filled with a variety of conducting or semiconducting materials.

A mass spectrometric approach to the study of DNA-binding proteins: interaction of human TRF2 with telomeric DNA.

Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.

Structural studies of the Maillard reaction products of a protein using ion trap mass spectrometry.

The early stage products of the Maillard reaction of egg white lysozyme with D-glucose were studied. Incubation with D-glucose at 50 degrees C for 20 days caused reaction on the Lys and Arg residues of lysozyme as follows: all of the six Lys residues and 10 of the 11 Arg residues in lysozyme reacted with D-glucose; Arg 61 did not react with D-glucose. The Lys residues reacted with D-glucose with 1 mol of dehydration per mole of residue, and the Arg residues reacted with 2 mol of dehydration per mole of residue. The major constituent of the Amadori product with the epsilon-amino group of the Lys residue and the D-glucose was found to be the beta-pyranose form. The structure of the early stage product of the Maillard reaction of a protein with a sugar is the same as that of an amino acid with a sugar.

Guidelines for reporting the use of capillary electrophoresis in proteomics

1Department of Comparative Molecular Medicine, School of Veterinary Science, The University of Liverpool, Liverpool, UK. 2Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, UK. 3Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, UK. 4National Institute for Biological Standards and Guidelines for reporting the use of column chromatography in proteomics

Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry.

The effect of hydroxyl radical induced oxidation on the collision cross-sections of hen egg lysozyme and bovine ubiquitin was investigated by travelling wave ion mobility mass spectrometry for the first time. The oxidized ions of lysozyme and ubiquitin share common collision cross-sections with their unoxidized counterparts suggesting that they share common structures that were unaffected by limited oxidation. In the case of bovine ubiquitin, two distinct conformers were detected for the protein in its unoxidized and oxidized states though no change in the levels of each was observed upon oxidation. This supports the validity of Radical Probe Mass Spectrometry (RP-MS) using an electrical discharge source for protein footprinting experiments. Travelling wave ion mobility mass spectrometry has been used for the first time to confirm that limited oxidation does not have an impact on the global structure of proteins.

Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure

Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination. Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution. Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament. Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination. In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.