Folding of the transcription factor Brinker and interactions of the bacterial second messenger c-di-GMP studied by NMR

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a technique, which allows the non-invasive investigation of structures, dynamics and interactions of biomolecules. The main goal of this thesis was to elucidate the folding mechanism of the transcription factor Brinker and its implications for DNA recognition as well as the characterization of unfolded protein states by NMR. This constitutes the first part of this thesis. The transcription factor Brinker is a nuclear repressor, which is involved in cellular growth and differentiation. In the absence of DNA, Brinker is completely disordered. However, in the presence of DNA or at low temperatures, the Brinker DNA binding domain (BrkDBD) adopts a well-folded structure. Thus, BrkDBD represents an extreme case of the coupling between binding and folding phenomenon. We have aimed to elucidate this folding mechanism in order to understand its implications for DNA recognition. From our data, it is clear that the BrkDBD folding energy landscape sharply depends on buffer anion type and concentration. We show that folded BrkDBD always adopts the same structure irrespective of the conditions. Our data indicate helical propensity for 3 of the 4 native helices even in unfolded BrkDBD, which may serve as initial contact points for DNA recognition. Resonance broadening due to conformational exchange on the micro- to millisecond time scale between folded and unfolded BrkDBD was analyzed by NMR relaxation dispersion experiments indicating a two-state folding mechanism. Only few residues show a different behavior and these are all located at the DNA binding interface. This local conformational heterogeneity may be important for DNA recognition. Based on these findings, we propose a mechanism of DNA recognition by BrkDBD, where the electrostatics-driven folding is a key component, accelerating the recognition process. In addition, we have analyzed the side-chain chi1-rotamer distribution of urea-denatured ubiquitin and protein G, revealing that individual residues show significant deviations from statistical-coil ensemble averages, indicating local bias towards the folded state. The second part of this thesis describes the quantitative characterization of the intermolecular interactions between monomers of the bacterial second messenger c-di-GMP at physiologically relevant concentrations. C-di-GMP is a bacterial second messenger, involved in many signaling events. Its most important effect is to trigger the transition from motile to sessile bacterial life-styles which plays a major role in biofilm formation. In solution, c-di-GMP has been reported to form several oligomers in the presence of monovalent cations, particularly potassium. However, only monomeric and dimeric c-di-GMP have been observed in complexes with proteins or RNA. We have carried out a detailed kinetic and thermodynamic analysis of c-di-GMP polymorphism in the presence of potassium, which showed that predominantly monomers and only few dimers exist at physiological concentrations. Additionally, we present NOE and ROE structural information on c-di-GMP oligomers, which indicate that these are not entirely all-syn and all-anti as opposed to the literature.