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.