This thesis concerns the structure and internal dynamics inDNA and RNA-binding proteins. While it has long been known thata proper three-dimensional structure is essential for aprotein's ability to interact with other biomolecules, theinfluence of the inherent protein dynamics is less wellunderstood. Using nuclear magnetic resonance (NMR)spectroscopy, both structure and dynamics within a protein canbe assessed. By applying two- and three-dimensional NMRtechniques on uniformly isotopically labelled protein samples,the solution structures of biomolecules or biomolecularcomplexes with molecular weights up to approximately 35 000 Dacan be determined. In addition, protein dynamics on manydifferent timescales can be investigated using severaldifferent experimental techniques, for example relaxationexperiments and amide hydrogen exchange measurements. Inparticular 15N relaxation measurements give a very useful viewof the protein mobility along the peptide backbone.
DNA/RNA-binding proteins participate in many processes inthe cell and there is still a lot to be learned about how theseproteins exert their function. A characterisation of theinternal dynamics in these proteins and if and how theirmobility influences the biological funcition is an importantstep in this work. This thesis involves the DNA-bindingproteins Sso7d fromSulfolobus solfatancusand the glucocorticoid receptorDNA-binding domain (GRDBD) as wellasthe ribosomal RNA binding protein S 15 fromThennus thermophilus.We have shown that thesequencespecific binding glucocorticoid receptor DNA-binding domain isa well ordered protein also in the uncomplexed state and thatGRDBD is not subjected to any major local folding processesupon DNA complexation. We could also show increased dynamics ina triple mutant with altered DNA binding specificity.Furthermore, arginine side chain dynamics was investigated indifferent protein surroundings; buried in a protein core inGRDBD, solvent-exposed on the Sso7d surface, and finally at theSso7d-DNA interface. We could demonstrate that the side chainmobility on the protein-DNA interface was restricted but to alesser extent than in the core region of a protein. Finally,NMR methods were used to determine the solution structure ofthe ribosomal rRNA binding protein S 15. The structure revealeda predorninandy ot-helical fold and allowed for identificationof a putative RNA-interacting surface.
Keywords:nuclear magnetic resonance spectroscopy,protein dynamics, nucleic acid binding proteins, NMR relaxationexperiments
Institutionen för biokemi och biokemisk teknologi , 1997. , 30 p.