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Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the μM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site. [ABSTRACT FROM AUTHOR]

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for 'artificial alpha repeat protein') have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal 'Velcro' to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself. [ABSTRACT FROM AUTHOR]

Diaphragmatic paralysis, a difficult diagnosis in the pediatric age group, has classically been made by fluoroscopy or B-mode ultrasound. We report our experience with TM-mode exploration. Twenty-seven patients suspected to have diaphragmatic paralysis were examined by means of inspiratory and expiratory chest radiography, fluoroscopy and B-mode ultrasound. The diaphragmatic echo was recorded on TM-tracing during spontaneous breathing using coronal oblique scans. Direction, excursion and the pattern of the transition between inspiration and expiration were analysed. In 7 patients examination was normal and TM mode demonstrated movement of normal direction and excursion with a sharp aspect of the transition zone. Diaphragmatic paralysis was present in 11 patients: unilateral in 9 and bilateral in 2 cases. TM mode demonstrated paradoxical movement, reduced excursion and a smooth transition zone. In 9 patients with diaphragmatic dysfunction TM mode demonstrated movement in the normal direction but with reduced excursion and a smooth transition zone. Compared to other imaging modalities, TM-mode records diaphragmatic movements more objectively. It can identify direction of the movement even if they are fast and of weak amplitude and in the case of bilateral paralysis. TM can differentiate paralysis from dysfunction. Moreover, this low-cost, non-irradiating made of imaging can be performed at the bedside and is available on all basic devices. [ABSTRACT FROM AUTHOR]

Artificial proteins binding any predefined "target" protein can now be efficiently generated using combinatorial libraries based on robust protein scaffolds. αRep is such a family of artificial proteins, based on an α-solenoid protein repeat scaffold. The low aggregation propensity of the specific "binders" generated from this library opens new protein engineering opportunities such as the creation of biosensors within multidomain constructions. Here, we have explored the properties of two new types of artificial bidomain proteins based on αRep structures. Their structural and functional properties are characterized in detail using biophysical methods. The results clearly show that both bidomain proteins adopt a closed bivalve shell-like conformation, in the ligand free form. However, the presence of ligands induces a conformational transition, and the proteins adopt an open form in which each domain can bind its cognate protein partner. The open/closed equilibria alter the affinities of each domain and induce new cooperative effects. The binding-induced relative domain motion was monitored by FRET. Crystal structures of the chimeric proteins indicate that the conformation of each constituting domain is conserved but that their mutual interactions explain the emergent properties of these artificial bidomain proteins. The ligand-induced structural transition observed in these bidomain proteins should be transferable to other αRep proteins with different specificity and could provide the basis of a new generic biosensor design. [ABSTRACT FROM AUTHOR]

Thymic hemorrhage related to coagulopathy by vitamin K deficiency is a rare condition. We report a case in a 4-week-old boy presenting with acute onset of respiratory distress and anemia. Ultrasound examination of a compressive anterior mediastinal mass established the diagnosis of thymic hemorrhage and allowed us to follow resolution under medical treatment. [ABSTRACT FROM AUTHOR]