Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters. The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface. PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC. The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media. For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values. In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments. / Ph. D.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.