Table of Contents:
Calcium-dependent protein kinases (CDPKs) form large gene families in plants
and various protists and it was proposed that members may be involved in
different signalling pathways.
The objective of this thesis was the detection of conformational changes of
NtCDPK2 from tobacco (Nicotiana tabacum) which were induced by external
stress stimuli. Because no structural data from crystallization or NMR
studies are available for full length CDPKs, three independent in vivo
methods were applied to investigate the conformational changes of NtCDPK2 in
planta. This information should also help to elucidate the biochemical
reaction mechanism of the enzyme during its in vivo activation in planta.
Using the split ubiquitin (SplitUB) system intermolecular interactions of CDPK
protein subdomains were first analysed in trans. The splitUB system
was then further developed for the study of in vivo protein domain
interactions in planta based on the Agrobacterium tumefaciens-mediated
transient expression in Nicotiana benthamiana. An in vivo interaction
between the calmodulin-like domain and the autoinhibitory junction domain of
NtCDPK2 could be demonstrated.
Furthermore, by investigating NtCDPK2 variants carrying point mutations in
specific domains a correlation between the conformational information
obtained by the SplitUB system in cis and the biological activity of the enzyme was observed. These conformational changes were shown to be dependent on kinase
function either by a mutation in the active centre of the kinase or by the
alteration of the structure of the autoinhibitory junction domain. In
addition, integrity of the Ca2+-binding EF-hands was required for NtCDPK2
Based on a second system, bimolecular fluorescence complementation, an
interaction of the N-terminal domains (VKJ) with the calmodulin-like domain
of NtCDPK2 could also be shown in N. benthamiana.
Third, intramolecular interactions indicating changes in the overall
conformations of the protein were analysed in planta using fluorescence
resonance energy transfer (FRET). Depending on the structural and thus
functional properties of the autoinhibitory domain of NtCDPK2, different
protein conformations could also be observed.
These data support and complement a previously proposed model for CDPK in
However, none of these systems allowed the detection of a fast kinetic of
significant conformational changes for NtCDPK2 directly triggered by
external stress stimuli in vivo.
In an independent approach the SplitUB system was applied to analyse
conformational changes of the UV-A/blue light receptor AtCRY2 from
Arabidopsis thaliana. This cryptochrome is proposed to become activated in a
blue light-dependent manner. In this thesis, in vivo evidence was obtained
for the first time showing a light-dependent conformational change of
cryptochrome based on the application of the SplitUB system in planta.