Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions
between DevS and DevR that are implicated in the regulation of the dormancy response in
Mycobacterium tuberculosis. These binding interactions could provide good drug targets for
the treatment of persistent tuberculosis, the mechanistic understanding of their binding
interactions is important for the development of a validated inhibitor screen. A detailed in
silico analysis of the amino acid residues that play a role in the binding of receptor DevR to
both kinase DevS and the target DNA was undertaken. A reasonable approximation of the
DevS structure was produced using homologous protein structures. In silico docking of DevS
to DevR merely produced a set of probable candidate structures, since more than one
conformation with similar docked energies was observed. The decision on which one is the
more correct form can only be estimated by crystallization of this complex. Therefore, the
functional expression and purification of the Dev TCS components were pursued. Denaturing
HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the
production of functional Dev TCS proteins. To understand the binding of DevR to DNA
consensus sequences, as well as the nature of these interactions, a model was built of the full
length DevR dimer binding to DNA consensus sequences. Based on this model, single
mutations were made to DevR in vitro and their effects assessed in order to validate the
model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that
K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences.
If DevR and DevS binding are to be used in a drug development program, it is essential to
have the protocols to accurately measure their interaction, in addition to developing a
fundamental understanding of how their interactions occur. The binding affinity of DevR to
both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the
BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a
histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was
calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded
that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore
could be used to screen small-molecule inhibitors of DevR-DevS interactions. / AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante
stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken
vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings
interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder
toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die
binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model
van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico
mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf,
aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die
mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie
kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik
uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel
affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik.
Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die
aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan
DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep
en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie
analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die
DNS konsensus volgordes.
Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die
fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die
interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om
die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te
ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD
in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS,
onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR
bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om
klein-molekule inhibeerders van DevR-DevS interaksies te toets.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20099 |
| Date | 03 1900 |
| Creators | Botha, Maria Magdalena |
| Contributors | Wiid, I. J. F., Kenyon, C. P., Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 136 p. : ill. |
| Rights | Stellenbosch University |
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