Distal unfolding of ferricytochrome C induced by the F82K mutation

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82\u2014conserved amino acid and a key actor in cytochrome c intermolecular interactions\u2014with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by1 H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization

Serum or Plasma (and Which Plasma), That Is the Question

Blood derivatives are the biofluids of choice formetabolomic clinical studies since blood can be collected with lowinvasiveness and is rich in biological information. However, the choiceof the blood collection tubes has an undeniable impact on the plasmaand serum metabolic content. Here, we compared the metabolomicand lipoprotein profiles of blood samples collected at the same timeand place from six healthy volunteers but using different collectiontubes (each enrolled volunteer provided multiple blood samples at adistance of a few weeks/months): citrate plasma, EDTA plasma, andserum tubes. All samples were analyzed via nuclear magnetic resonancespectroscopy. Several metabolites showed statistically significantalterations among the three blood matrices, and also metabolites'correlations were shown to be affected. The effects of blood collectiontubes on the lipoproteins'profiles are relevant too, but less marked. Overcoming the issue associated with different blood collectiontubes is pivotal to scale metabolomics and lipoprotein analysis at the level of epidemiological studies based on samples frommulticenter cohorts. We propose a statistical solution, based on regression, that is shown to be efficient in reducing the alterationsinduced by the different collection tubes for both the metabolomic and lipoprotein profile

Oblique stimulated Raman scattering of a short laser pulse in a plasma channel

The spatiotemporal evolution of parametric instabilities such as stimulated Raman scattering is studied analytically in time and two spatial dimensions. Initial and boundary conditions are chosen to represent the entrance, propagation, and exit of a laser pulse of finite extent as it progresses through a homogeneous collisional plasma channel. For most scattering angles daughter wave growth is enhanced by lateral reflections within the channel. At late times the two-dimensional interaction approaches a one-dimensional damped mode in which the dissipative loss from lateral transmission of the Stokes wave through the channel boundaries is equivalent to an overall damping of the Stokes amplitudes within the channel

Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin

Adsorption of the prototypical organic corrosion inhibitor benzotriazole on the Cu(100) surface

The interaction of benzotriazole (BTAH) with Cu(100) has been studied as a function of BTAH exposure in a joint experimental and theoretical effort. Scanning tunnelling microscopy (STM), X-ray photoelectron spectroscopy (XPS), high resolution electron energy loss spectroscopy (HREELS) and density functional theory (DFT) calculations have been combined to elucidate the structural and chemical characteristics of this system. BTAH is found to deprotonate upon adsorption on the copper surface and to adopt an orientation that depends on the molecular coverage. Benzotriazolate (BTA) species initially lie with their planes parallel to the substrate but, at a higher molecular coverage, a transition occurs to an upright adsorption geometry. Upon increasing the BTAH exposure, different phases of vertically aligned BTAs are observed with increasing molecular densities until a final, self-limiting monolayer is developed. Both theory and experiment agree in identifying CuBTA and Cu(BTA)2 metal-organic complexes as the fundamental building blocks of this monolayer. This work shows several similarities with the results of previous studies on the interaction of benzotriazole with other low Miller index copper surfaces, thereby ideally completing and concluding them. The overall emerging picture constitutes an important starting point for understanding the mechanism for protection of copper from corrosion

Diauxie and co-utilization of carbon sources can coexist during bacterial growth in nutritionally complex environments

It is commonly thought that when multiple carbon sources are available, bacteria metabolize them either sequentially (diauxic growth) or simultaneously (co-utilization). However, this view is mainly based on analyses in relatively simple laboratory settings. Here we show that a heterotrophic marine bacterium, Pseudoalteromonas haloplanktis, can use both strategies simultaneously when multiple possible nutrients are provided in the same growth experiment. The order of nutrient uptake is partially determined by the biomass yield that can be achieved when the same compounds are provided as single carbon sources. Using transcriptomics and time-resolved intracellular 1H-13C NMR, we reveal specific pathways for utilization of various amino acids. Finally, theoretical modelling indicates that this metabolic phenotype, combining diauxie and co-utilization of substrates, is compatible with a tight regulation that allows the modulation of assimilatory pathways

Modelling hCDKL5 heterologous expression in bacteria

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production