A detailed atomistic knowledge of protein-protein and protein-RNA interactions is indispensable for an understanding of the biochemical function of these important chemical entities. In the project we apply our recently developed integrated NMR methodologies for structural biology studies of RNA-binding proteins and protein-RNA complexes using an interdisciplinary approach combining NMR spectroscopy, novel bioinformatic tools, molecular biology, and bioorganic chemistry. Specifically, in the frame of the SFB the focus of our research will be the efficient sequence-based bioinformatic analyses of proteins and capitalizing on the derived features the fast and automated protein structure determination, analysis of protein and RNA dynamics, and applications to protein-RNA interaction studies.

Solution NMR studies of Hfq and Hfq-RNA complexes
We have successfully completed the NMR determination of the solution structure of Hfq. It was unambiguously shown that Hfq exists as a hexamer in solution. The overall shape of the protein oligomer is very similar to the crystal structure. Although previous attempts to reconstitute a Hfq/RNA complex were not successful, we will continue with these experiments using full-length Hfq and different RNA constructs. Of particular interest is the question whether conformational dynamics across the monomer interfaces influence RNA recognition by optimizing and fine tuning intermolecular interactions between nucleic acids and residues from Hfq. As much of protein function is predicated on dynamics, we anticipate that these experiments may help to unravel the microscopic details of Hfqs structural plasticity and its relevance for function. NMR was also used to provide information about the RNA binding site. Additionally, the development of a novel NMR technique allowed for the determination of the oligomerization state of Hfq in solution at very low concentration.

Bioinformatic screening and comprehensive sequence-analysis of RNA-binding and RNA chaperoning proteins
For sequence-based protein structural analyses a new bioinformatic tool was developed (The Meta-Structure Approach) which provides information about residue exposure and secondary structure elements. This novel prediction tool is used to set up a large-scale in silico screen to identify new RNA binding proteins/chaperones by screening against a validated database of about 100 RNA chaperone sequences. Additionally, by including sequence-specific biochemical information (e.g. residues involved in RNA binding) significant sequence markers for chaperone activity are likely to be established. Additionally the tools can be used to identify potential ligand binding sites exclusively based on the RNA nucleotide sequence information.

In vitro NMR interaction and structural studies of RNA-binding (chaperoning) proteins
RNA can typically fold into several isoenergetic structures, giving place to misfolded and non-functional structures. It is known that proteins can assist in RNA folding by two different mechanisms: binding and stabilizing specific structures, or by what is called RNA chaperone activity (RCA), an activity that accelerates folding through the resolution of misfolded structures or inhibition of their formation. Therefore one goal is to gain insight into RNA chaperone function on an atomic level. We use CspA as a model of RNA chaperone. CspA is the major cold shock protein in E. coli. It is a 70 residues protein that serves to induce adaptation to cold shock conditions (10-15°). Previous studies in our group on CspA dynamics showed that the protein is 1% unfolded at 32°C (M. Tollinger, unpublished results), it has single-stranded nucleic acid binding capacity. It is supposed to act as transcriptional anti-terminator for cold shock genes. We have already determined the solution structure of CspA (both in the unligated state and when bound to RNA) and the location of the nucleic acid binding site. Preliminary NMR studies indicate that CspA assists and accelerates duplex formation of RNA hairpins (presumably due to unfolding of RNA secondary structures).
NMR studies of RNA conformational dynamics
To extend the applicability of NMR spectroscopy to nucleic acid structural biology we have recently started a cooperation with the group of Ronald Micura (University of Innsbruck) combining synthetic RNA chemistry and 19F NMR spectroscopy to probe conformational states and binding events in larger RNAs. We have shown that a 2’-F atom can efficiently mimic the 2’-OH group and thus does not significantly disturb structurally important hydrogen bonding interactions. Given the exquisite dependence of the 19F chemical shifts on the structural and electronic environment it is an excellent and sensitive reporter of RNA conformational properties. Additionally, we have presented a novel concept for the identification and subsequent analysis of RNA-binders by 19F NMR spectroscopy. The methodology was demonstrated with applications to a tobramycin RNA aptamer and Mg2+-induced binding of flavine mononucleotide to in vitro selected RNA bulge motifs. In the frame of the SFB these novel tools will be very valuable to study both structural and dynamical features of RNAs both in their unligated forms and when bound to their respective targets.