CONICET | Buscador de Institutos y Recursos Humanos

The advent of structural genomics have fostered an increasing interest in the development of NMR methods that provide atom-resolved structural information on proteins in short overall time using sensitive NMR pulse schemes. Previously, several longitudinal relaxation optimized experiments for proteins were introduced that allow for a substantial reduction in acquisition time and for a correlated increase in signal to noise ratio per unit time1,2. Reduced NMR data acquisition times become a crucial issue whenever the molecular system under investigation has a short lifetime in the NMR sample tube. It is also important for high-throughput NMR applications in the context of structural genomics initiatives. We present here the application of these experiments to the screening of protein constructs and a new set of optimized liquid crystal NMR experiments for the obtention of RDC restraints. The protein fragments amenable to structural characterization by NMR are first identified from small-scale high-throughput purified samples taking advantage of the enhanced sensitivity. Then RDC datasets are acquired on the selected constructs and used directly to calculate the protein fold using the Rosetta NMR software. Processing of microRNA in plants involves several multidomain proteins. The procedure outlined above was applied to characterize structurally the RNA binding domains of HYL13 and DCL1 from A. thaliana and their interaction with RNA substrates. REFERENCES: Schanda P and Brutscher B, J. Am. Chem. Soc. 127:8014-8015 (2006). Lescop E et al. J. Magn. Reson. 187:163-169 (2007). Rasia et al., Biochemistry 49:82378239 (2010)