Human serum transferrin (hTF) is a bilobal glycoprotein that plays a central role in iron metabolism. Each lobe of hTF (N- and C-lobe) can reversibly bind a single ferric iron. Iron binds to hTF at neutral pH in the plasma; diferric hTF binds to specific hTF receptors (TFR) on the cell surface and the complex undergoes receptor mediated endocytosis. The pH within the endosome is lowered to ~5.6 and iron is released from hTF. Apo hTF remains bound to the TFR and recycles back to the cell surface. Upon fusion with the plasma membrane, apo hTF dissociates from the TFR and is free to bind more iron and continue the cycle. The iron release process is complicated by various factors which include pH, anions, a chelator, lobe-lobe cooperativity and interaction with the TFR. All of these influence iron release in a complex manner. Because they are intricately linked, it is difficult to determine the effect of any single parameter. We have utilized stopped-flow and steady-state fluorescence and urea gel electrophoresis to dissect the iron release process as a function of lobe-lobe interactions, the presence of the TFR, and changes in pH and salt concentration. Application of recombinant protein production and site-directed mutagenesis has allowed us to generate a variety of hTF constructs in which the iron status of each lobe is completely controlled. Thus, we have created authentic monoferric hTFs unable to bind iron in one lobe, diferric hTFs with iron locked in one lobe and diferric hTF in which iron can be removed from both lobes. Importantly, we have produced the soluble portion of the TFR (sTFR) to analyze interactions between hTF and the sTFR and to monitor iron release from hTF/sTFR complexes. Together, we are able to provide a more precise picture of iron release from the two lobes of hTF in the presence and absence of the TFR. Steady-state fluorescence emission scans and urea gel electrophoresis provide a qualitative evaluation of the iron status of each construct after a predetermined incubation in iron removal buffer (i.e. an endpoint). However, these techniques do not provide information regarding the kinetic pathway to reach that endpoint. Combined with stopped-flow fluorescence time-based kinetics, a more precise assessment of the iron release process has been obtained. We have determined that changes in pH and salt affect endpoint iron release from the C-lobe, but not the N-lobe, however, the kinetics of iron release from both lobes are highly sensitive to pH and salt. Kinetic analysis in the absence and presence of the sTFR reveals the complexity of the iron release process. In the absence of the sTFR, the kinetics of iron release are insensitive to the iron status of the opposite lobe. However, in the presence of the sTFR, the kinetics of iron release from both lobes are affected by the iron status of the opposite lobe. Determination of conformational changes induced by anion binding, lobe-lobe communication and sTFR interactions have now been confidently assigned. We have created kinetic models of iron release from diferric hTF ± the sTFR and incorporated specific events pertaining to anion binding, lobe-lobe communication and conformational changes associated with sTFR interactions. We provide irrefutable evidence that a critical role of the sTFR is to accelerate the rate of iron release from the C-lobe, while decreasing the rate of iron release from the N-lobe such that the two lobes effectively release iron on a time scale relevant to one cycle of endocytosis.

Studying protein interaction networks using functional and topological information is important for understanding cellular organization and functionality. This study deals with identifying important proteins in protein interaction networks using SWEMODE (Lubovac, et al, 2006) and analyzing topological and functional properties of these proteins with the help of information derived from modular organization in protein interaction networks as well as information available in public resources, in this case, annotation sources describing the functionality of proteins. Multi-modular proteins are short-listed from the modules generated by SWEMODE. Properties of these short-listed proteins are then analyzed using functional information from SGD Gene Ontology(GO) (Dwight, et al., 2002) and MIPS functional categories (Ruepp, et al., 2004). Topological features such as lethality and centrality of these proteins are also investigated, using graph theoretic properties and information on lethal genes from Yeast Hub (Kei-Hoi, et al., 2005). The findings of the study based on GO terms reveal that these important proteins are mostly involved in the biological process of “organelle organization and biogenesis” and a majority of these proteins belong to MIPS “cellular organization” and “transcription” functional categories. A study of lethality reveals that multi-modular proteins are more likely to be lethal than proteins present only in a single module. An examination of centrality (degree of connectivity of proteins) in the network reveals that the ratio of number of important proteins to number of hubs at different hub sizes increases with the hub size (degree).

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2?m. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.

Iron is an essential element for virtually all forms of life. Complicating matters, it is present in the insoluble ferric form in aerobic environments, while the more soluble ferrous form is found in anaerobic or reducing environments. Vibrio cholerae, the causative agent of the disease cholera, requires iron to survive. In order to meet the need for iron, V. cholerae expresses a variety of iron acquisition systems. One of these systems, Feo, is highly conserved among bacterial species as well as archaea and transports ferrous iron. The Feo system consists of three proteins: FeoA, FeoB, and FeoC. Previous work using the bacterial adenylate cyclase two hybrid system has shown that FeoC interacts with the cytoplasmic N-terminal domain of FeoB. However, the significance of this interaction is not known. In this study, V. cholerae Feo system proteins were analyzed for residues important for the interaction between FeoB and FeoC. In addition, FeoA and FeoC were purified for antibody production. It was found that a residue in the G protein domain of FeoB was not necessary for interaction with FeoC. However, a conserved residue in FeoC did abolish the interaction with FeoB. These results indicate that there is at least one residue important in the interaction of FeoB and FeoC, although further characterization will most likely reveal more. Antibodies to FeoA and FeoC were generated to use them for further characterization of the Feo system. / text

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason

ABSTRACT The ability to detect and respond to environmental signals is fundamental in coordinating floral induction in plants to favourable conditions. An important flowering time cue is day length and it is proposed that light signals are perceived and measured by an interaction between photoreceptors and an internal pacemaker, the circadian clock. The control of flowering has been best characterised in the model plant Arabidopsis thaliana L. Heynh (Arabidopsis). The GIGANTEA (GI) gene has a complex role in both the promotion of flowering in response to photoperiod and the regulation of the circadian clock. The expression of GI is under circadian control and is affected by day length, light quality and temperature changes. The GI protein is also circadian regulated and is actively degraded in the dark. The biochemical function of GI is unknown and one method to elucidate the role of this protein is to identify protein interactors. The aim of this thesis project was to characterise proteins that interacted with GI. Previously, the BELL-LIKE HOMEODOMAIN 3 (BLH3) protein was identified as a putative GI protein interactor. As part of this thesis work, yeast 2-hybrid and in vitro pull down assays were utilised to confirm the interaction between GI and BLH3. Sequence and phylogenetic analyses were used to further examine the BELL family of proteins. The BELL-LIKE HOMEODOMAIN 10 (BLH10) protein was found to be closely related to BLH3 and also interacted with GI. Reverse 2-hybrid assays were used to determine the regions or domains within the GI, BLH3 and BLH10 proteins required to mediate protein interactions. Expression assays established that the BLH3 and BLH10 transcripts were present throughout plant tissues and times of development. Further analyses revealed that BLH3 and BLH10 are not directly regulated by the circadian clock. The results of GFP expression assays demonstrated that the BLH3 protein is localised to the nucleus in plant cells. Transgenic blh3 and blh10 mutant plants were identified and analysed for flowering and light response phenotypes. BLH3 and BLH10 do not function with GI in the photoperiodic pathway to control flowering, yet the blh3 and blh10 mutants do have a flowering phenotype in short day conditions. Like gi, the blh3 and blh10 mutants exhibited exaggerated hypocotyl elongation in response to red and low light conditions. These results are suggestive of a role for BLH3, BLH10 and GI in flowering and deetiolation responses to specific light conditions in plants. / This PhD research was sponsored by Dr George Mason