Cytochromes c7 are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins - phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.

Protein phosphorylation is a complex and highly dynamic process involved in numerous biological events. Abnormal phosphorylation is one of the underlying mechanisms for the development of cancer and metabolic disorders. The identification and absolute quantification of specific phospho-signatures can help elucidate protein functions in signaling pathways and facilitate the development of new and personalized diagnostic and therapeutic tools. This review presents a variety of strategies currently utilized for the enrichment of phosphorylated proteins and peptides before mass spectrometry analysis during proteomic studies. The investigation of specific affinity reagents, allied to the integration of different enrichment processes, is triggering the development of more selective, rapid and cost-effective high-throughput automated platforms.

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The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol−1 for GSU0935 and ΔG = 26.3 kJ.mol−1 for GSU0582), and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582), which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding and stability are at the basis of the specificity and regulation of many types of PAS-containing signaling proteins.

The identification and localization of the proteinaceous binders are essential issues in studies of painting materials and techniques, for further proposing valid restoration and conservation treatments of the painted or polychrome works of art. The challenge for analytical chemists and conservation scientists is the availability of methods able to simultaneously identify and map the presence of the binders in the multilayered structure of a sample and the possibility to use a very low amount of sample from the studied art object (considering also the criteria of minimum sampling). These methods should be fast, reproducible in different artefacts and in case of mixture of protein-based binders with other non-proteinaceous constituents (oils, resins, waxes, gums etc.) and also economical (both in terms of materials and time consume). In this context, the present paper proposes an innovative protocol of investigation using two complementary techniques – Matrix-Assisted Laser Desorption/Ionisation – Time of Flight Mass Spectrometry (MALDI-TOF MS) and staining tests (one visible and one fluorescent stain) assisted by Optical Microscopy (OM) on cross-section of samples – for the simultaneous identification and mapping of protein – and oil-based binders in paint materials. The novelty is based on the use of MALDI-TOF MS on cross-sections of paints together with a fluorescent stain for protein identification and mapping (mainly used in the area of proteomics) complementing the use of a traditional visible stain for oil-based material identification. The protocol was successfully applied on several samples taken from a Czech medieval polychrome sculpture, entitled “The Mourning of Jesus Christ” (16th century) belonging to the Moravian Gallery (Brno).

Cloud computing has been dominated by system-level virtual machines to enable the management of resources using a coarse grained approach, largely in a manner independent from the applications running on these infrastructures. However, in such environments, although different types of applications can be running, the resources are often delivered in a equal manner to each one, missing the opportunity to manage the available resources in a more efficient and application aware or driven way. Our proposal is QoE-JVM supporting Java applications with a global and elastic distributed image of a high-level virtual machine (HLLVM), where total resource consumption and allocation (within and across applications in the infrastructure) are driven by incremental gains in quality-of-execution (QoE), which relates the resources allocated to an application and the performance the application can extract from having those resources. In this paper, we discuss how critical resources (memory and CPU) can be allocated among HLL-VMs, so that Cloud providers can exchange resource slices among virtual machines, continually adaptdressing where those resources are required, while being able to determine where the reduction will be more economically effective, i.e., will contribute in lesser extent to performance degradation.

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Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co-factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co-factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co-factors.