Descripción del proyecto
Espectroscopia por RMN de grandes complejos proteicos
La espectroscopia por resonancia magnética nuclear (RNM) permite identificar la dinámica proteica de una disolución en una amplia variedad de escalas temporales y con resolución atómica. Sin embargo, los estudios con RMN de proteínas grandes siguen planteando dificultades debido a que los métodos convencionales de RMN se limitan a proteínas pequeñas. El proyecto financiado con fondos europeos NMRofLargeComplexes tiene por objeto ampliar los límites de la RMN hasta los grandes complejos proteicos mediante la combinación de desarrollos recientes en el marcado de puntos específicos mediante nuevos métodos de RMN y espectrometría. Este desarrollo facilitará el estudio de dinámicas importantes desde el punto de vista funcional e interacciones con el sustrato de grandes complejos proteicos, ofreciendo información sobre la relación entre la dinámica, la estructura y la función. Los estudios actuales se centrarán en el exosoma eucariota, un decámero asimétrico soluble de 370 kDa, y el antiportador NapA Na+/H+ de 80 kDa, un dímero de membrana integral implicado en el transporte del hierro.
Objetivo
Dynamic properties of proteins are essential for their function. NMR can reveal protein dynamics in solution on a broad range of timescales and at atomic resolution. However, NMR studies of large proteins remain challenging since conventional NMR methods are constrained to small proteins. The aim of this project is to extend the NMR size limit to large protein complexes by combining recent developments in site-specific labeling schemes with novel NMR methods employing state-of-the-art spectrometers. This will facilitate the study of functionally essential but so far uncharacterized dynamics and substrate interactions of two large protein complexes, providing unprecedented insight into the relationship between dynamics, structure and function. Since most proteins are dynamic, extending the scope of protein complexes that are amenable to NMR study is of great interest to many areas of molecular biology. Studies will be conducted on the eukaryotic exosome, a 370 kDa soluble, asymmetric decamer involved in RNA degradation and processing, and the 80 kDa Na+/H+ antiporter NapA, an integral membrane dimer involved in ion transport. For the exosome, individual subunits will be 13C-methyl labeled; the employment of sophisticated NMR methods will then permit the detection of functionally essential dynamics and RNA-substrate binding patterns of the exosome. As NapA is a low-yield membrane protein, an inexpensive 19F-labeling approach will be employed to reveal global transport dynamics and local gating motions. Exosome production and all NMR experiments are to be conducted in Remco Spranger’s lab at the University of Regensburg, Germany. NapA will be produced during a secondment in David Drew’s lab at Stockholm University, Sweden. This newly established international collaboration will permit integration of methods and knowledge to study protein systems previously inaccessible to NMR, strengthen the profile of the applicant and foster research of the involved institutes.
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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinador
93053 Regensburg
Alemania