NMR studies of biomolecular complexes

Solution heteronuclear NMR spectroscopy was used toward structural characterization of the RNA binding domains of the protein, Sex-lethal (Sxl). These domains have a common conserved sequence and structural motif referred to as the ribonucleoprotein motif (RNP). The backbone resonances of the first domain of Sxl (Sxl-RBD1), and those of the two domain protein (Sxl-RBD1+2) were assigned. Chemical shift perturbations upon Sxl-RBD1+2 binding of cognate RNA were compared in the two domains, and analyzed for differences relative to other structurally characterized RNP type RNA binding proteins. Orientational restraints were obtained for Sxl-RBD1, Sxl-RBD2, Sxl-RBD1+2, and Sxl-RBD1+2 plus RNA. These restraints were used to analyze the relative orientation of the two domains in Sxl-RBD1+2 when bound to RNA and support a model that has the RNA binding faces oriented towards one another. Work towards improving the accuracy of this orientational information is discussed.; Solution proton NMR was used to determine the structure of a 22 residue DNA aptamer in complex with argininamide. The DNA folds to form a hairpin structure with five basepairs in the stem, and ten residues in the loop. The basepair at the top of the stem, and eight of the ten residues in the loop are critical for DNA binding of argininamide. The DNA in complex contains several non-canonical base pairs in the loop that are only formed upon ligand binding. This structure also exhibits a unique binding pocket that is formed in part by unusual out of sequence looping by one of the DNA bases resulting in a large kink in the backbone of the loop. This kink allows the argininamide to be encapsulated by the DNA. Formation of this complex also exhibits fast to intermediate exchange binding kinetics that are analyzed using NMR lineshape theory to determine information on the binding constant and kinetics of the system.