Biomedical subjects

Dana E Robinson

Publications and source records attributed to Dana E Robinson.

5 recordsLinked to original sources

PubMed2006

Evolutionary migration of a post-translationally modified active-site residue in the proton-pumping heme-copper oxygen reductases.

In the respiratory chains of aerobic organisms, oxygen reductase members of the heme-copper superfamily couple the reduction of O2 to proton pumping, generating an electrochemical gradient. There are three distinct families of heme-copper oxygen reductases: A, B, and C types. The A- and B-type oxygen reductases have an active-site tyrosine that forms a unique cross-linked histidine-tyrosine cofactor. In the C-type oxygen reductases (also called cbb3 oxidases), an analogous active-site tyrosine has recently been predicted by molecular modeling to be located within a different transmembrane helix in comparison to the A- and B-type oxygen reductases. In this work, Fourier-transform mass spectrometry is used to show that the predicted tyrosine forms a histidine-tyrosine cross-linked cofactor in the active site of the C-type oxygen reductases. This is the first known example of the evolutionary migration of a post-translationally modified active-site residue. It also verifies the presence of a unique cofactor in all three families of proton-pumping respiratory oxidases, demonstrating that these enzymes likely share a common reaction mechanism and that the histidine-tyrosine cofactor may be a required component for proton pumping.

Amino Acid Sequence↗

PubMed2006

Top down mass spectrometry of < 60-kDa proteins from Methanosarcina acetivorans using quadrupole FRMS with automated octopole collisionally activated dissociation.

A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5x increase in experimental throughput and a 2x increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5-59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies.

Amino Acid Sequence↗

PubMed2005

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism.

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).

Aminoacyltransferases↗

PubMed2005

Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry.

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high sequence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of human proteins (and their possible variants) was created and searched using tandem mass spectrometric data from intact proteins. This first application of top down MS/MS to wild-type human proteins demonstrates both gene-specific identification and the unambiguous characterization of multifaceted mass shifts (Deltam values). Such Deltam values found from the precise identification of 45 protein forms from HeLa cells reveal 34 coding single nucleotide polymorphisms, two protein forms from alternative splicing, and 12 diverse modifications (not including simple N-terminal processing), including a previously unknown phosphorylation at 10% occupancy. Automated protein identification was achieved with a median expectation value of 10(-13) and often occurred simultaneously with dissection of diverse sources of protein variability as they occur in combination. Top down MS therefore has a bright future for enabling precise annotation of gene products expressed from the human genome by non-mass spectrometrists.

Alternative Splicing↗

PubMed2004

Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

Acetylation↗