About the author Ana Garcia del Arco

5ª Reunión científica del consorcio Tec4Bio

5ª Reunión científica del consorcio Tec4Bio

Con la participación de todos los grupos miembros del Consorcio Tec4Bio-CM, celebramos el pasado jueves 5 de maup nuestra 5ª reunión científica del programa, en esta ocasión acudieron además los miembros del comité evaluador externo. ¡Gracias a todos por vuestra participación!

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CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

Marina Serna, Ana González-Corpas, Sofía Cabezudo, Andrés López-Perrote, Gianluca Degliesposti, Eduardo Zarzuela, J Mark Skehel, Javier Muñoz and Oscar Llorca

Abstract: Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1–RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1–RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.

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Molecular Recognition by Silicon Nanowire Field-Effect Transistor and Single-Molecule Force Spectroscopy

Francisco M. Espinosa, Manuel R. Uhlig and Ricardo Garcia.

Abstract: Silicon nanowire (SiNW) field-effect transistors (FETs) have been developed as very sensitive and label-free biomolecular sensors. The detection principle operating in a SiNW biosensor is indirect. The biomolecules are detected by measuring the changes in the current through the transistor. Those changes are produced by the electrical field created by the biomolecule. Here, we have combined nanolithography, chemical functionalization, electrical measurements and molecular recognition methods to correlate the current measured by the SiNW transistor with the presence of specific molecular recognition events on the surface of the SiNW. Oxidation scanning probe lithography (o-SPL) was applied to fabricate sub-12 nm SiNW field-effect transistors. The devices were applied to detect very small concentrations of proteins (500 pM). Atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS) experiments allowed the identification of the protein adsorption sites on the surface of the nanowire. We detected specific interactions between the biotin-functionalized AFM tip and individual avidin molecules adsorbed to the SiNW. The measurements confirmed that electrical current changes measured by the device were associated with the deposition of avidin molecules.

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Accurate Wide-Modulus-Range Nanomechanical Mapping of Ultrathin Interfaces with Bimodal Atomic Force Microscopy

Victor G. Gisbert and Ricardo Garcia

Abstract: The nanoscale determination of the mechanical properties of interfaces is of paramount relevance in materials science and cell biology. Bimodal atomic force microscopy (AFM) is arguably the most advanced nanoscale method for mapping the elastic modulus of interfaces. Simulations, theory, and experiments have validated bimodal AFM measurements on thick samples (from micrometer to millimeter). However, the bottom-effect artifact, this is, the influence of the rigid support on the determination of the Young’s modulus, questions its accuracy for ultrathin materials and interfaces (1–15 nm). Here we develop a bottom-effect correction method that yields the intrinsic Young’s modulus value of a material independent of its thickness. Experiments and numerical simulations validate the accuracy of the method for a wide range of materials (1 MPa to 100 GPa). Otherwise, the Young’s modulus of an ultrathin material might be overestimated by a 10-fold factor.

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