Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 μM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes

Language-Related Brain Potentials in Patients With Disorders of Consciousness: A Follow-up Study to Detect “Covert” Language Disorders

Background. Language disorders may occur in patients with disorders of consciousness (DoCs), and they could interfere with the behavioral assessment of consciousness and responsiveness. Objective. In this study, we retrospectively explored whether ERP N400 was eventually associated with the presence of aphasia diagnosed in those patients who had evolved into Exit-Minimally Conscious State (E-MCS) at the clinical follow-up. Methods. In this retrospective cohort study, the ERPs elicited by an auditory sentences task were retrospectively examined in a sample of 15 DoC patients diagnosed according to the Coma Recovery Scale–Revised (CRS-R). All these 15 DoC patients underwent a (at least) 1-year clinical follow-up, which included a neuropsychological evaluation to assess language function among other cognitive functions. Ten healthy individuals also underwent the same paradigm to investigate the variability of ERPs characteristics. Results. The N400 ERP component with centroparietal topography was found in 9 of 10 healthy controls in response to the ill-formed sentences. Analysis of patients’ data revealed that (1) a significant N400 component could be detected in 64% (9 of 14 patients) of the DoC patients regardless of the type of DoC; (2) no significant N400 ERP component was retrospectively detected in those E-MCS patients who showed aphasia at the follow-up; and (3) the presence/absence of the N400-ERP component was consistent with the brain lesion side and significantly predict the recovery. Conclusion. These preliminary findings indicate that the absence of N400 was significantly associated with the presence of aphasia diagnosed at the clinical follow-up in E-MCS patients

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe–4S] cluster

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CXnCCGXmCXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe–2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), 57Fe electron–nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe–4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe–4S]2+ cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with gzyx = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with gzyx = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S3(O/N)1 coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron–sulfur cluster and the zinc site

Intrinsic Fluorescence of Intrinsically Disordered Proteins

Resolution of the intrinsic emission properties of a protein by different fluorescence spectroscopy techniques is an invaluable tool to detect and characterize its structural architecture and conformational changes under different experimental conditions. Indeed, the multidimensional character of fluorescence can provide information on local chemical features, on solvent diffusional processes, and on rotational movements of peptide chains or whole proteins. Here, we describe the details of quenching fluorescence experiments and how to correlate the results to the peculiar structural information on the organization of intrinsically disordered proteins (IDPs)

Esame del Linguaggio (II versione)

Organizzazioni Speciali Firenz

Let not the left hand know what the right hand knoweth

Following a cerebral vascular accident, a patient showed a classical disconnection syndrome: left-hand tactile anomia, apraxia and dysgraphia and right-hand constructional apraxia. What made the case unusual was the presence of hand asymmetry in the performance of some matching-to-sample tasks carried out in foveal vision. The left hand committed significantly more errors than the right hand when it was not possible to identify on a perceptual basis the stimulus that was to be matched, because it was removed (memory condition) or was indicated verbally (verbo-visual matching), or had the same name but not the same physical appearance as the match (capital and lower-case letter matching). No hand difference emerged when the stimulus remained in full view throughout the matching task (perceptual condition). The hand effect, however, was limited to colours and letters. Objects, geometrical shapes and unfamiliar faces were matched with equal proficiency by both hands under every condition of presentation. Left-hand errors also significantly outnumbered right-hand errors in sorting colours according to hue and colouring drawings. MRI showed an infarct in the left cingulate white matter that ran parallel to the trunk of the corpus callosum, and an infarct of the splenium. However, the latter did not prevent the transmission of colour and letter information between the two hemispheres, as shown by the performance on perceptual equivalence tasks and by the correct right-hand responses to stimuli projected to the left visual field. We propose that this pattern of deficit is contingent upon the specific role that the left hemisphere plays in categorizing a given colour patch as belonging to a definite colour region (red, blue, etc.) and in grapheme recognition. Without the assistance of the left side the right hemisphere lacks the benefit provided by meaning identification. In our patient the left brain did receive information from the right brain and was able to process it, but was prevented by the paracallosal lesion from transmitting what it knew to the right motor area. No hand effect emerged for objects and geometrical shapes, because their semantic memory is bilaterally represented

Esame del Linguaggio (II versione).

Organizzazioni Speciali Firenz

Denaturant-Induced Conformational Transitions in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) differ from ordered proteins at several levels: structural, functional, and conformational. Amino acid biases also drive atypical responses of IDPs to changes in their environ- ment. Among several specific features, the conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding. In fact, the denaturant- induced unfolding of native molten globules can be described by shallow sigmoidal curves, whereas urea- or guanidinium hydrochloride-induced unfolding of native pre-molten globules or native coils is a noncoop- erative process and typically is seen as monotonous feature-less changes in the studied parameters. This chapter describes some of the most characteristic features of the IDP conformational behavior