A study of biomolecular interactions using three biological complexes to explore structure, dynamics and method development

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dc.contributor.advisorJohnson, Philip E.
dc.contributor.advisorDonaldson, Logan
dc.creatorBoodram, Sherry Nicole
dc.date.accessioned2016-08-03T16:51:34Z
dc.date.available2016-08-03T16:51:34Z
dc.date.copyright2013-08
dc.degree.disciplineChemistry
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractThe protein, RDE-4 in C. elegans, served as a model for studying how double stranded RNA binding proteins that bind to dsRNA molecules are critical for RNAi cellular processes. NMR spectroscopy confirms that the RDE-4 construct has characteristic protein domains that bind to. dsRNA and that RNA binding causes a significant global change of the protein structure. SAXS analysis indicates that the two binding domains in the RDE-4 protein do not interact with one another, but instead forms a continuous interface onto which long target dsRNA can bind. Gel shift assay experiments reveal that multiple RDE-4 molecules bind to a non-sequence specific RNA substrate with positive cooperativity. RNA binding occurs with micromolar affinity and a second binding event occurs with millimolar affinity. The binding of E. coli dihydrofolate reductase (DHFR) to inhibitors methotrexate (MTX) and 1,4-Bis-{[ N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (inhibitor 1) has been studied to investigate the dynamics involved in the catalytic mechanism of DHFR. NMR relaxation methods show that in the presence of inhibitor 1, the catalytic domain of DHFR binds 1 in the substrate-binding pocket and an occluded conformation is assumed. In both the DHFR:NADPH and DHFR:NADPH:1 complexes, motion is exhibited on the microsecond-millisecond timescale. The heat capacity change of DHFR upon binding to 1 and MTX are 43 ± 10 cal/mol-K and -120 ± 109 cal/mol-K respectively. Differences in ΔCp of DHFR binding to inhibitor 1 compared to that of MTX indicate that the mode of binding to 1 is different from what is observed in the crystal structure of the complex. An affinity electrophoresis method to screen for RNA-small molecule ligand interactions has been developed. This method is made quantifiable by cross-linking the ligand into the gel matrix and gauging binding by RNA mobility. The utility of this method is demonstrated using the known interaction between different aminoglycoside ligands with the E. coli ribosomal A-site RNA and with an RNA molecule containing a C-C mismatch. Average apparent dissociation constants are determined. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.
dc.identifier.urihttp://hdl.handle.net/10315/31690
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subject.keywordsBiomolecular interactions
dc.subject.keywordsBiological complexes
dc.subject.keywordsRDE-4 protein
dc.subject.keywordsRNA
dc.subject.keywordsRNAi cellular processes
dc.subject.keywordsdsRNA
dc.subject.keywordsE. coli dihydrofolate reductase
dc.subject.keywordsDHFR
dc.titleA study of biomolecular interactions using three biological complexes to explore structure, dynamics and method development
dc.typeElectronic Thesis or Dissertation

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