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1.
J Mol Biol ; 407(1): 149-70, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21262234

ABSTRACT

Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1+F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1+F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair.


Subject(s)
DNA Breaks, Single-Stranded , DNA/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Chromatin/metabolism , DNA Repair , Electrophoretic Mobility Shift Assay , Humans , Magnetic Resonance Spectroscopy , Mutation/genetics , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Protein Binding , Zinc Fingers
2.
Mol Cell Proteomics ; 9(8): 1774-83, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20467040

ABSTRACT

The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution.


Subject(s)
Phylogeny , Ribosomes/chemistry , Ribosomes/genetics , Tandem Mass Spectrometry , Animals , Archaea/metabolism , Eukaryota , Molecular Weight , Ribosomal Proteins/chemistry , Temperature , Thermus thermophilus/growth & development , Thermus thermophilus/metabolism
3.
J Mol Biol ; 380(2): 404-14, 2008 Jul 04.
Article in English | MEDLINE | ID: mdl-18514735

ABSTRACT

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in approximately 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Protein Subunits/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Acetylation , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Molecular Weight , Protein Processing, Post-Translational , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomes/chemistry
4.
J Biol Chem ; 283(5): 2595-603, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18055467

ABSTRACT

Proton-translocating ATPases are central to biological energy conversion. Although eukaryotes contain specialized F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases but simpler in design. Although the nucleotide-binding and transmembrane rotor subunits share sequence homology between A-, V-, and F-ATPases, the peripheral stalk is strikingly different in sequence, composition, and stoichiometry. We have analyzed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display-derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A(1) domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes.


Subject(s)
Bacterial Proton-Translocating ATPases/chemistry , Bacterial Proton-Translocating ATPases/metabolism , Thermus thermophilus/enzymology , Animals , Antibodies, Bacterial , Bacterial Proton-Translocating ATPases/genetics , Bacterial Proton-Translocating ATPases/immunology , Base Sequence , DNA, Bacterial/genetics , Microscopy, Immunoelectron , Models, Molecular , Multiprotein Complexes , Peptide Library , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Tandem Mass Spectrometry , Thermus thermophilus/genetics
5.
Mol Cell Proteomics ; 6(7): 1135-46, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17322308

ABSTRACT

Protein synthesis in mammalian cells requires initiation factor eIF3, an approximately 800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a-m) with a sequence coverage of approximately 79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex.


Subject(s)
Eukaryotic Initiation Factor-3/chemistry , Protein Biosynthesis , Protein Processing, Post-Translational , Chromatography, Liquid , Eukaryotic Initiation Factor-3/metabolism , HeLa Cells , Humans , Phosphorylation , Protein Subunits/chemistry , Protein Subunits/metabolism , Tandem Mass Spectrometry/methods
6.
Proc Natl Acad Sci U S A ; 102(23): 8192-7, 2005 Jun 07.
Article in English | MEDLINE | ID: mdl-15923259

ABSTRACT

Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.


Subject(s)
Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Thermus thermophilus/metabolism , Amino Acid Sequence , Bacillus subtilis/chemistry , Escherichia coli/chemistry , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Molecular Weight , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Ribosomal Protein L10 , Ribosomal Proteins/chemistry , Static Electricity , Thermus thermophilus/chemistry , Thermus thermophilus/cytology
7.
FEBS Lett ; 579(4): 943-7, 2005 Feb 07.
Article in English | MEDLINE | ID: mdl-15680979

ABSTRACT

The ability to maintain intact ribosomes in the mass spectrometer has enabled research into their changes in conformation and interactions. In the mass spectrometer, it is possible to induce dissociation of proteins from the intact ribosome and, in conjunction with atomic structures, to understand the factors governing their release. We have applied this knowledge to interpret the structural basis for release of proteins from ribosomes for which no high resolution structures are available, such as complexes with elongation factor G and ribosomes from yeast. We also describe how improvements in technology and understanding have widened the scope of our research and lead to dramatic improvements in quality and information available from spectra of intact ribosomes.


Subject(s)
RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Spectrometry, Mass, Electrospray Ionization , Ions/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism
8.
Biophys J ; 88(3): 2114-25, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15626715

ABSTRACT

The surfactant properties of aqueous protein mixtures (ranaspumins) from the foam nests of the tropical frog Physalaemus pustulosus have been investigated by surface tension, two-photon excitation fluorescence microscopy, specular neutron reflection, and related biophysical techniques. Ranaspumins lower the surface tension of water more rapidly and more effectively than standard globular proteins under similar conditions. Two-photon excitation fluorescence microscopy of nest foams treated with fluorescent marker (anilinonaphthalene sulfonic acid) shows partitioning of hydrophobic proteins into the air-water interface and allows imaging of the foam structure. The surface excess of the adsorbed protein layers, determined from measurements of neutron reflection from the surface of water utilizing H(2)O/D(2)O mixtures, shows a persistent increase of surface excess and layer thickness with bulk concentration. At the highest concentration studied (0.5 mg ml(-1)), the adsorbed layer is characterized by three distinct regions: a protruding top layer of approximately 20 angstroms, a middle layer of approximately 30 angstroms, and a more diffuse submerged layer projecting some 25 angstroms into bulk solution. This suggests a model involving self-assembly of protein aggregates at the air-water interface in which initial foam formation is facilitated by specific surfactant proteins in the mixture, further stabilized by subsequent aggregation and cross-linking into a multilayer surface complex.


Subject(s)
Proteins/chemistry , Proteins/ultrastructure , Ranidae/metabolism , Surface-Active Agents/chemistry , Water/chemistry , Adsorption , Air , Animals , Multiprotein Complexes/analysis , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Protein Binding , Proteins/analysis , Surface Tension , Surface-Active Agents/analysis
9.
J Biol Chem ; 279(41): 42750-7, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15294894

ABSTRACT

The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.


Subject(s)
Mass Spectrometry/methods , Ribosomal Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Blotting, Western , Gene Deletion , Ions , Isoelectric Focusing , Methylation , Mutation , Phosphoric Monoester Hydrolases/chemistry , Phosphorylation , Ribosomes/chemistry , Ribosomes/metabolism , Spectrometry, Mass, Electrospray Ionization
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