Structural Biology without Structure - Delta Subunit of  RNA Polymerase from Bacillus subtilis

 

Lukáš Žídek

 

1NCBR and CEITEC, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

lzidek@chemi.muni.cz

 

Intrinsically disordered proteins are currently attracting an increased interest of biochemists and structural biologists.  A (partial) structural disorder has been observed in a considerable portion of human proteome, including proteins related to serious diseases. Unstructured proteins present a particular challenge for structural biology, as the standard techniques utilizing X-ray diffraction or NMR distance data fail to describe the disordered portions of protein molecules at atomic level. Yet, various methods can be applied to describe conformational behaviour of the unstructured polypeptide chains. Several NMR techniques were used to characterize the unstructured C-terminal domain of the delta subunit of Bacillus subtilis RNA polymerase. Non-standard data acquisition and processing methods have been developed to overcome very poor resolution of the NMR spectra of the delta subunit. The possibility to distinguish all signals in the spectra allowed us to map the local conformation reflected by the bias of resonance frequencies from their random-coil values. Flexibility of the disordered polypeptide was described in detail by NMR relaxation measurements. Paramagnetic labels were introduced to reveal transient contacts, beyond the limit of the nuclear Overhauser effect. The average overall shape of the molecule was studied by analytical ultracentrifugation. Combination of the obtained data provided a very detailed description of the C-terminal region, complementing the high-resolution structure of the well-ordered N-terminal domain.

 

Acknowledgements.

This work has been supported by the project "CEITEC - Central European Institute of Technology" from European Regional Development Fund, grant number CZ.1.05/1.1.00/02.0068 and by grants P206/11/0758, 204/09/0583, and  301/09/H004 from the Czech Science Foundation. Access to the Bio-NMR Research Infrastructure co-funded under the 7th Framework Programme of the EC (FP7/2007-2013), grant number 261863 for conducting the research is gratefully acknowledged.