1H, 13C, 15N resonance assignment of human YAP 50-171 fragment.

Yes associated protein (YAP) is an intrinsically disordered protein that plays a major role in the Hippo pathway, regulating organ size, cell proliferation, apoptosis, and is associated with cancer development. Therefore, the binding between YAP and TEAD is an interesting target for cancer therapy. The TEAD binding domain of YAP was mapped to protein residues 50-171. To obtain further structural insights into this 12 kDa segment of YAP, we report a backbone and a partial sidechain assignment of recombinant YAP 50-171.

Lung recruitment prevents collapse during laparoscopy in children: A randomised controlled trial.

BACKGROUND: Capnoperitoneum and anaesthesia impair lung aeration during laparoscopy in children. These changes can be detected and monitored at the bedside by lung ultrasound (LUS). OBJECTIVE: The aim of our study was to assess the impact of general anaesthesia and capnoperitoneum on lung collapse and the potential preventive effect of lung recruitment manoeuvres, using LUS in children undergoing laparoscopy. DESIGN: Randomised controlled study. SETTING: Single-institution study, community hospital, Mar del Plata, Argentina. PATIENTS: Forty-two children American Society of Anesthesiologists I-II aged 6 months to 7 years undergoing laparoscopy. INTERVENTIONS: All patients were studied using LUS before, during and after capnoperitoneum. Children were allocated to a control group (C-group, n=21) receiving standard protective ventilation, or to a lung recruitment manoeuvre group (RM-group) (n=21), in which lung recruitment manoeuvres were performed after recording baseline LUS images before capnoperitoneum. Loss of aeration was scored by summing a progressive grading from 0 to 3 assigned to each of 12 lung areas, based on the detection of four main ultrasound patterns: normal aeration = 0, partial loss-mild = 1, partial loss-severe = 2, total loss-consolidation = 3. MAIN OUTCOME MEASURES: Lung aeration score and atelectasis assessed by ultrasound. RESULTS: Before capnoperitoneum and recruitment manoeuvres in the treated group the two groups presented similar ultrasound scores (5.95 ±â€Š4.13 vs. 5.19 ±â€Š3.33, P = 0.5). In the RM-group, lung aeration significantly improved both during (2.71 ±â€Š2.47) and after capnoperitoneum (2.52 ±â€Š2.86), compared with the C-group (6.71 ±â€Š3.54, P < 0.001, and 8.48 ±â€Š3.22, P < 0.001, respectively). There was no statistically significant difference in the percentage of atelectasis before capnoperitoneum and recruitment manoeuvres in the RM-group (62%) and in the C-group (47%, P = 0.750). However, during capnoperitoneum, only 19% of the RM-group had atelectasis compared with 80% in the C-group (P < 0.001). CONCLUSION: The majority of children undergoing laparoscopy have anaesthesia-induced atelectasis. In most cases, lung collapse due to capnoperitoneum could have been prevented by recruitment manoeuvres followed by positive-end expiratory pressure. TRIAL REGISTRY NUMBER: NCT02824146.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

Network representation of protein interactions-Experimental results.

A graph theoretical analysis of nuclear magnetic resonance (NMR) data of six different protein interactions has been presented. The representation of the protein interaction data as a graph or network reveals that all of the studied interactions are based on a common functional concept. They all involve a single densely packed hub of functionally correlated residues that mediate the ligand binding events. This is found independent of the kind of protein (folded or unfolded) or ligand (protein, polymer or small molecule). Furthermore, the power of the graph analysis is demonstrated at the examples of the Calmodulin (CaM)/Calcium and the Cold Shock Protein A (CspA)/RNA interaction. The presented approach enables the precise determination of multiple binding sites for the respective ligand molecules.

Novel approaches in selective tryptophan isotope labeling by using Escherichia coli overexpression media.

NMR-based investigations of large protein complexes require optimized isotopic labeling schemes. We report new methods to introduce stable isotopes into tryptophan residues; these are fine-tuned to the requirements of the particular protein NMR experiment. Selective backbone labeling was performed by using a new α-ketoacid precursor as an additive in cell-based overexpression media. Additionally, we developed synthetic routes to certain isotopologues of indole with (13)C-(1)H spin systems surrounded by (12)C and (2)H. The corresponding proteins, overexpressed in the presence of these precursor compounds, can be effectively analyzed for conformational changes in tryptophan residues in response to external stimuli, such as interaction with other proteins or small molecules.

Magnetic resonance access to transiently formed protein complexes.

Protein-protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein-protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins.

Strategies for purifying variants of human rhinovirus 14 2C protein.

The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.