| dc.contributor.advisor | Fritz, Hans-Joachim Prof. Dr. | de |
| dc.contributor.author | Ramakrishnan, Venkatesh | de |
| dc.date.accessioned | 2012-05-16T12:08:17Z | de |
| dc.date.available | 2013-01-30T23:50:44Z | de |
| dc.date.issued | 2005-07-29 | de |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0006-B5FA-A | de |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-1282 | |
| dc.format.mimetype | application/pdf | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://webdoc.sub.gwdg.de/diss/copyr_diss.html | de |
| dc.title | Structural Analysis of a Transactivation Domain Cofactor Complex | de |
| dc.type | doctoralThesis | de |
| dc.title.translated | Structural Analysis of a Transactivation Domain Cofactor Complex | de |
| dc.contributor.referee | Griesinger, Christian Prof. Dr. | de |
| dc.date.examination | 2005-06-30 | de |
| dc.subject.dnb | 500 Naturwissenschaften allgemein | de |
| dc.description.abstracteng | Signal Transducers and Activators of Transcription
(STAT) proteins were discovered as targets for
interferon activation. These proteins are dormant in
the cytoplasm until they are activated by one or more
cytokines, that interact with their cognate cell
surface receptors. These extracellular signaling
proteins can activate various tyrosine kinases inside
the cell that phosphorylate the STAT proteins. The
activated STAT proteins migrate to the nucleus and
drive transcription. Since STAT activity is modulated
by several post-translational modifications and
protein-protein interactions, these transcription
factors are capable of integrating inputs from multiple
signaling networks.The carboxy-terminal region of the
STAT proteins functions as a transcriptional activation
domain. Seven mammalian STATs have been discovered so
far. Inspite of the progress that has been made in
identifying the interaction targets of the activation
domains and the recognition of the importance of
particular residues for their function, the structural
basis for the ability of transactivation domains to
stimulate transcription is not understood well. The
structural understanding of the transactivation domains
is barely developed, in contrast to the well known
DNA-binding domains. This thesis work involved the
biochemical and structural characterization of the
interaction between the transactivation domain of STAT6
and its co-factor, the Nuclear Receptor Co-activator
-1(NCoA-1). The amino acid sequences in both STAT6 and
NCoA-1 involved in this interaction had been identified
previously.The sequence spanning amino acids 794-814 in the
STAT6 C- terminal region was identified to be essential
in mediating the interaction with NCoA-1 and comprised
of a LXXLL motif. In the case of NCoA-1, the region
between the amino acids 213-462 had been identified to
be involved in the interaction. The complex was
reconstituted in vitro. Biochemical characterization of
this complex using techniques like gel filtration,
limited proteolysis, mass spectroscopy and protein
sequencing suggested that the residues 257-385 of the
NCoA-1 is the minimal domain required for the
interaction with the STAT6 and that the ratio of the
binding partners in the complex was 1:1. Secondary
structure prediction showed that the region C- terminal
to residue 366 in the NCoA-1 was unstructured. Two
shorter constructs of NCoA-1, one spanning residues
257-385 and other comprising of residues 257-370 were
cloned for further experiments. Titration calorimetry
showed that the dissociation constant of the binding
was in the order of 10?7M. The crystal structure of the
complex between the NCoA-1(257?385) and STAT6(794?814)
peptide revealed that the NCoA-1(257?385) fragment in
complex with STAT6(794?814) peptide forms a canonical
Per-Arnt-Sim (PAS) domain. The free forms of both the
NCoA-1 constructs failed to crystallize, therefore NMR
was used for the study of these constructs. Isotope
labeled NCoA-1(257?385) and NCoA-1(257?370) fragments
were used for multidimensional NMR experiments.
Backbone resonance assignmentproved that the residues
C- terminal to amino acid 366 were unstructured.Further structural analysis using Residual Dipolar
Couplings (RDCs) was not successful, as the couplings
obtained were not suitable for such studies. The reason
was that the alignment of the protein in liquid
crystalline medium which is a pre-requisite for the
measurement of dipolar couplings was not optimal. | de |
| dc.subject.topic | Mathematics and Computer Science | de |
| dc.subject.ger | Transaktivation | de |
| dc.subject.ger | Struktural | de |
| dc.subject.ger | NMR | de |
| dc.subject.eng | Transactivation | de |
| dc.subject.eng | Structural | de |
| dc.subject.eng | NMR | de |
| dc.subject.bk | Biologie | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-webdoc-105-9 | de |
| dc.identifier.purl | webdoc-105 | de |
| dc.affiliation.institute | Biologische Fakultät inkl. Psychologie | de |
| dc.subject.gokfull | WCC 000: Molekulare Biophysik. Biophysikalische Chemie | de |
| dc.identifier.ppn | 497824884 | de |