Structural Studies of Flexible Biomolecules and a DNA-binding Protein

Abstract: The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute ?,? Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide ?-D-Mannosep-(1-2)-?-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the ?,? distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate ?,? distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent ?-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at ?H=-40° ?H=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ?H angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid ?-helices made up by residues 5-66 and a ?-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.