Biological physics: harmonies from noise.
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Alleles↗
Biomedical subjects
Michael Springer
Publications and source records attributed to Michael Springer.
Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.
Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.
Chemistry Techniques, Analytical↗
A distal, high-affinity binding site on the cyclin-CDK substrate Pho4 is important for its phosphorylation and regulation.
Cyclins and cyclin-dependent kinases (CDKs) are key components of signaling pathways essential for cell growth and survival. The cyclin-CDK Pho80-Pho85 inactivates the transcription factor Pho4 in budding yeast by phosphorylating it on five sites. We isolated seven single amino acid substitutions outside of the phosphorylation sites that cause Pho4 to be constitutively active. The substitutions decrease the amount of Pho4 phosphorylation in vivo, and they increase the apparent K(M) of the in vitro phosphorylation reaction by an order of magnitude but do not alter k(cat) substantially. These data suggest that the substituted residues are part of a cyclin-CDK-binding site that is distal to the phosphorylation sites. Further analysis revealed that all of Pho4 variants were phosphorylated by Pho80-Pho85 in a more distributive manner than the wild-type protein, further supporting the idea that binding at a distal, high-affinity binding site is important in determining the processivity of Pho4 phosphorylation. In addition, computational modeling of the Pho4 phosphorylation reactions shows that the K(D) of binding between the Pho4 mutants and Pho80-Pho85 increases, confirming that the mutations are located in a relatively high-affinity "docking site" for the kinase. Interestingly, the K(D) derived from the in vitro data correlates well with the strength of the in vivo phenotypes, demonstrating that the in vitro data are relevant to the in vivo regulation of Pho4.
Amino Acid Substitution↗
Viral discovery and sequence recovery using DNA microarrays.
Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.
Base Sequence↗
Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes.
A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.
Blotting, Northern↗