Chemical & Structural Biology

Comparison of the NMR Spectroscopy Solution Structure of Pyranosyl-RNA and Its Nucleo-delta-peptide Analogue

For all biological systems, nature has chosen ribo- and deoxyribonucleic acids as its genetic building block. In order to understand this selectivity, the structures of the potential alternatives to the natural nucleic acids have to be investigated. We have determined the solution structures of pRNA and Nucleo-d-peptides by NMR spectroscopy. The structures pose important questions about the origin of helicity, stacking and inclination of these oligomers.


Fig 2: Structure of pRNA and NDP (Ilins et al., ChemBioChem 2001, in press; Schwalbe et al., Helv. Chim. Acta 83, 1079-1107 (2000)) in collaboration with groups of Prof. Quinkert (Univ. Frankfurt), Prof. Eschenmoser and Prof. Jaun (ETH Zürich).



NMR spectroscopy is used to determine the structure of proteins, of RNA and DNA in order to provide structural insight into intermolecular interactions.


Fig: Structure of calmodulin complexed with the target peptide C20W from the Ca-ATPase pump (Elshorst et al., Biochemistry 38, 12320-12332 (1999)) in collaboration with groups of Prof. Griesinger (MPI Göttingen) and Prof. Carafoli (ETH Zürich).

NMR studies of the structure and dynamics of the ribosomal protein L11 from Thermotoga maritima.

L11 is highly conserved ribosomal protein and its interaction with the ribosomal RNA segment from 23S subunit is considered to undergo an „induced fit“-conformational adjustment of both the protein and the RNA with respect to their conformations in the unbound state (Leulliot and Varani, 2001; Draper et al., 1996). Furthermore, the conformational dynamics of L11 are thought to play an important role in the binding process. Binding of the C-terminal domain of L11 stabilizes the tertiary structure of a compactly folded RNA domain whereas the N-terminus is implicated in the binding to the antibiotic thiostrepton (Xing and Draper, 1996). The conformation of the RNA-L11-complex is structurally well characterized (Wimberly et al., 1999, Conn et al., 1999). In addition, the conformation of the C-terminal domain of L11 from Bacillus stearothermophilus has been characterized in its RNA bound and free form by NMR (Hinck et al., 1997, Markus et al., 1997). Yet, to obtain a more detailed picture of the dynamic processes accompanying RNA-protein interactions we characterize in detail the conformation and the dynamics of the full-length protein free in solution. In particular, the relative orientation of the N- and C-terminal domain of the protein in its free form is being investigated by NMR-spectroscopy. By analysis of long-range structural information derived from heteronuclear relaxation rates and residual dipolar couplings, it can be shown that the two domains are connected by a relatively rigid poly-Proline helix, their relative flexibility is low and the prominent conformation in solution preorganizes the correct conformation in complex with RNA.


Overlay of the backbones of the 20 best structures out of 50. a) Backbone atoms of residues 5-70 that define N-terminus were fit by a least-square method. The r.m.s.d. for this superimposition is 0.28 Ċ for backbone atoms and 0.77 Ċ for all heavy atoms. b) Backbone atoms of resides 75-140 that define C-terminus. The r.m.s.d. for the superimposition of the well defined regions 97-111 and 121-140 is 0.295 Ċ for the backbone atoms and 0.82 Ċ for all heavy atoms. The beta-sheets and alpha-helices are color-coded and their first and last residues are indicated.

Development of New NMR Techniques for Monitoring of Protein Folding with Atomic Resolution

We have developed two independent methods to study structural transitions and reactions by time-resolved NMR spectroscopy with millisecond dead-time. Therefore, on one hand, we use a rapid mixing system inside the active volume in the probe, which we developed in cooperation with BRUKER. In addition, we have developed a method to laser-trigger cofactor dependent reactions inside the NMR tube which we established. These studies are supported by a wide variety of other spectroscopic techniques including stopped-flow fluorescence, CD, FTIR and EPR to gain further insight in structural characteristics during the folding process. At the moment we are focusing on the structural characterization of transient intermediates in protein folding. We are studying the kinetics of the natural structural changes of a-lactalbumine and calmoduline upon ion addition in cellular conditions, the binding kinetics of calmoduline to the calcium-pump receptor peptides and the refolding from denaturating reagents of calmodulin, a-lactalbumine, lysozyme and ubiquitin.


Fig 4: Experimental setup for the study of light induced reactions by high resolution NMR. (Till Kühn and Harald Schwalbe, J. Am. Chem. Soc. 122, 6169-6174 (2000); Wirmer et al., 2001 submitted).

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