Biomedical subjects

Thomas Taverner

Publications and source records attributed to Thomas Taverner.

4 recordsLinked to original sources

PubMed2006

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗

PubMed2006

Structural organization of the 19S proteasome lid: insights from MS of intact complexes.

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex.

COP9 Signalosome Complex↗

PubMed2003

Gas phase ion chemistry of charged silver(I) adenine ions via multistage mass spectrometry experiments and DFT calculations.

Electrospray ionization (ESI) of solutions containing adenine and AgNO(3) yields polymeric [Ad(x)+ Ag(y)-zH]((y-z)+) species. Density functional theory (DFT) calculations have been used to examine potential structures for several of the smaller ions while multistage mass spectrometry experiments have been used to probe their unimolecular reactivity (via collision-induced dissociation (CID)) and bimolecular reactivity (via ion-molecule reactions with the neutral reagents acetonitrile, methanol, butylamine and pyridine). DFT calculations of neutral adenine tautomers and their silver ion adducts provide insights into the binding modes of adenine. We find that the most stable [Ad + Ag](+) ion does not correspond to the most stable neutral adenine tautomer, consistent with previous studies that have shown that transition metal ions can stabilize rare tautomeric forms of nucleobases. Both the charge and the stoichiometry of the [Ad(x)+ Ag(y)-zH]((y-z)+) complexes play pivotal roles in directing the types of fragmentation and ion-molecule reactions observed. Thus, [Ad(2)+ Ag(2)](2+) is observed to dissociate to [Ad + Ag](+) and to react with butylamine via proton transfer, while [Ad(2)+ Ag(2)- H](+) fragments via loss of neutral adenine to form the [Ad + Ag(2)- H](+) ion and does not undergo proton transfer to butylamine. DFT calculations on several isomeric [Ad(2)+ Ag(2)](2+) ions suggest that planar centrosymmetric cations, in which two adjacent silver atoms are bridged by two N7H adenine tautomers via N(3),N(9)-bidentate interactions, are the most stable. The [Ad + Ag(2)-H](+) ion adds two neutral reagents in ion-molecule reactions, consistent with the presence of two vacant coordination sites. It undergoes a silver atom loss to form the [Ad + Ag - H](+) radical cation, which in turn fragments quite differently to the even electron [Ad + Ag](+) ion. Several other pairs of radical cation/even electron adenine-silver complexes were also found to undergo different fragmentation reactions.

Journal Article↗

PubMed2002

Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry.

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6(D) were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 (15)N-labeled and unlabeled ((14)N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [(15)N/(15)N] + [(15)N/(14)N] + [(14)N/(15)N] + [(14)N/(14)N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [(15)N] + [(14)N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).

Dimerization↗