FROM GENE TO 3D FOLD USING AUTOMATED CLONING, LABELING AND FAST NMR METHODS: APPLICATION TO THE STUDY OF PLANTS MICR

RASIA, R., BOLOGNA, N., NOIRCLERC-SAVOYE, M., GALLET, B., LESCOP, SCHANDA, P.,E., VERNET, T., BRUTSCHER, B., PALATNIK, J., BOISBOUVIER, J.

Brisbane, Australia

Congreso; HFSP annual meeting; 2007

HFSP

Liquid state NMR is a well-established method to solve the structure of small proteins. It can also be directly applied to larger proteins, provided they are modular and their corresponding domains fold independently. One of the time limiting steps in such approach is the determination of boundaries of structured domains. In order to efficiently study complex modular systems, we have developed a three-step protocol, which allows us to go from the cDNA to the protein fold.First, we have set up a fast and automated protocol to clone different truncated forms of the protein. These constructs are analyzed by NMR in order to determine minimal independently folded constructs.Once optimal folded constructs have been selected, the resonance frequencies of backbone nuclei for each amino-acid can be identified using newly developed experiments, which shortens the typical spectrometer time requirements from ~ 1 week to hours. These assignments of resonance frequencies can then directly be applied to the determination of residues involved in interactions with physiological partners. Finally, optimized liquid crystal NMR experiments and analysis tools have been developed for fast extraction of structural restraints. These data sets can be used directly for fold validation or structural refinements. Several multidomain proteins are known to participate in microRNA processing in plants. Here we show an application of this procedure to the structural characterization of the multidomain protein Hyl-1 from A. thaliana, a key component of the microRNA processing machinery in plants.