Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value ⤠0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 µM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.
Project description:Caenorhabditis elegans pup-2, PAP-associated domain-containing protein [Source:UniProtKB/TrEMBL;Acc:Q09408], is differentially expressed in 7 experiment(s);
Project description:Caenorhabditis elegans pup-3, PAP-associated domain-containing protein [Source:UniProtKB/TrEMBL;Acc:P91361], is differentially expressed in 3 experiment(s);
Project description:Caenorhabditis elegans pup-3, PAP-associated domain-containing protein [Source:UniProtKB/TrEMBL;Acc:P91361], is expressed in 1 baseline experiment(s);
Project description:Caenorhabditis elegans pup-2, PAP-associated domain-containing protein [Source:UniProtKB/TrEMBL;Acc:Q09408], is expressed in 1 baseline experiment(s);
Project description:We have performed in solution hydrogen exchange (HDX) on peptide standards and bovine hemoglobin with subsequent quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for the benchmarking of a lab built HDX-MS platform. Though neutral coated capillaries that eliminate electroosmotic flow provide fast separations with peak capaci-ties surpassing 200, uncoated capillaries with high electroosmotic flow achieve half as much peak ca-pacity with even faster separations but 30% higher deuterium retention for angiotensin II peptide standard. The data obtained from two different separation conditions on peptic digests of bovine he-moglobin shows strong agreement for relative deuterium uptake between methods and provides data amenable to software such as MS-Studio. Processed data from denatured hemoglobin vs the longest time point in this study (50,000s) also shows agreement with subunit interaction sites determined by crystallographic methods.
Project description:Hydrogen/deuterium exchange mass spectrometric methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in the deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.
Project description:Defining specific pathways for efficient heat transfer from protein-solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the application of hydrogen deuterium exchange coupled to mass spectrometry toward the spatial resolution of protein motions that can be related to the chemical reaction within an enzyme active site. Employing the proton-coupled electron transfer reaction of soybean lipoxygenase as proof of principle, we first corroborate the impact of active site mutation on increased local flexibility and second, uncover a solvent-exposed loop, 15-34 Å from the reactive ferric center whose temperature-dependent motions are demonstrated to mirror the enthalpic barrier for catalytic C-H bond cleavage. A network that connects this surface loop to the active site is structurally identified and supported by changes in kinetic parameters that result from site-specific mutations.