FlgM gains structure in living cells

Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (≥400 g/liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions

Concurrent recording of the electrically-evoked compound action potential and the auditory brainstem response in cochlear implant users

Purpose and Technological Feasibility • In the ABR waveform, WI reflects activity in the 8th cranial nerve while WV reflects activity in the upper brainstem. The amplitude ratio between WI and WV is emerging as an important metric for auditory function. • In the electrically evoked ABR (eABR), eWI cannot be measured because of artifact associated with cochlear implant (CI) stimulation. The electrical eWI /eWV ratio therefore cannot be measured in a single test as the WI /WV ratio can for an acoustically evoked ABR. • For MED-EL Corporation CIs, the electrically evoked compound action potential (eCAP), equivalent to eWI, is measured with the Auditory nerve Response Telemetry (ART) test. The ART uses sequences of ‘masker’ and ‘probe’ biphasic pulses to extract an eCAP (see Fig. 1). Note that the ART also generates an external trigger

Removal of a Wire Brush Bristle from the Hypopharynx Using Suspension, Microscope, and Fluoroscopy

Wire brush bristles are an increasingly recognized hazard that can present as a foreign body in the aerodigestive tract. Due to their small size and tendency to become embedded in surrounding tissue, these small metallic bristles present a unique operative challenge to otolaryngologists. Here we present a case of a 40-year-old woman who underwent endoscopic extraction of a wire bristle from the posterior pharyngeal wall using suspension, microscopy, and C-arm fluoroscopy. We believe this is the first published case of an endoscopic removal of a buried foreign body in the hypopharynx using these methods of localization concurrently. By leveraging multiple techniques for visualization, surgeons can avoid open exploration while ensuring complete removal of the object. Additionally, this case highlights the importance of regulatory oversight and consumer awareness of the hazards of grill brushes

Field Survey of Cucumber mosaic virus Subgroups I and II in Crop Plants in Costa Rica

copyright 2001 American Phytopathology Society Journals. Datos y artículo incluido por Lisela Moreira Carmona, responsable de depósitos de publicaciones del área de Patógenos y Plagas del CIBCMLeaf samples were collected from cucurbit and solanaceous crop plants and Musa spp. in 28 locations in five provinces of Costa Rica during the period from January to October 1996. Sampling sites were selected in dry, humid, and moist tropical regions ranging in altitude from 50 to 2,100 m above sea level. RNA-enriched total nucleic acid solutions were spotted onto nylon membranes and hybridized to RNA probes specific for Cucumber mosaic virus (CMV) subgroups I or II. The presence of CMV was confirmed in 13 crops in 23 of the 28 sampling sites. CMV subgroup I was found to predominate in Costa Rica. CMV subgroup II was detected in the Atlantic region only, and in only 1 out of 113 CMV-positive samples.Universidad de Costa Rica/[801-94-905]/UCR/Costa RicaUnited States Agency for International Development/[HRN-5600-G-00-2013-00]/USAD/Costa RicaUniversidad de Costa Rica/[801-95-583]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Atomic force microscopy based nanoassay: A new method to study \u3b1-Synuclein-dopamine bioaffinity interactions

Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular \u3b1-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of \u3b1-Synuclein on gold to study the early stages of \u3b1-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-\u3b1-Synuclein adducts

Intrinsically Disordered Proteins Display No Preference for Chaperone Binding In Vivo

Intrinsically disordered/unstructured proteins (IDPs) are extremely sensitive to proteolysis in vitro, but show no enhanced degradation rates in vivo. Their existence and functioning may be explained if IDPs are preferentially associated with chaperones in the cell, which may offer protection against degradation by proteases. To test this inference, we took pairwise interaction data from high-throughput interaction studies and analyzed to see if predicted disorder correlates with the tendency of chaperone binding by proteins. Our major finding is that disorder predicted by the IUPred algorithm actually shows negative correlation with chaperone binding in E. coli, S. cerevisiae, and metazoa species. Since predicted disorder positively correlates with the tendency of partner binding in the interactome, the difference between the disorder of chaperone-binding and non-binding proteins is even more pronounced if normalized to their overall tendency to be involved in pairwise protein–protein interactions. We argue that chaperone binding is primarily required for folding of globular proteins, as reflected in an increased preference for chaperones of proteins in which at least one Pfam domain exists. In terms of the functional consequences of chaperone binding of mostly disordered proteins, we suggest that its primary reason is not the assistance of folding, but promotion of assembly with partners. In support of this conclusion, we show that IDPs that bind chaperones also tend to bind other proteins

Self-Organization and Regulation of Intrinsically Disordered Proteins with Folded N-Termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism

In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-Synuclein within E. coli cells

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson’s disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution

In-cell NMR in E. coli to Monitor Maturation Steps of hSOD1

In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes

The Effect of a ΔK280 Mutation on the Unfolded State of a Microtubule-Binding Repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer's disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a ΔK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and ΔK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of β-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates