UNT Libraries - 2 Matching Results

Search Results

Designing Tools to Probe the Calcium-dependent Function of Arabidopsis Tonneau2

Description: Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function of TONNEAU at the PPB?” To address this question, a series of constructs were generated to determine if TON2 binds calcium. Additionally, Ca2+ binding sites were mutated in constructs containing the TON2 gene fused to GFP or YPF. These constructs were then transformed into ton2 mutant plants and the localization of TON2 fusion protein and whether the construct is capable of rescuing the mutant phenotype were observed. Although, localization of TON2 to the PPB was not observed, the presence of the constructs were confirmed in the transformed plants using selection markers and by observing fluorescence under a confocal microscope.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: December 2013
Creator: Oremade, Oladapo O.

Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2

Description: Hypertrophic cardiomyopathy (HCM), a heart-related abnormality, is the most prevalent cause of sudden death in young athletes at sporting events. A cluster of cardiomyopathy mutations are localized in β-cardiac myosin at the N-terminal region of subfragment-2. Using resonance energy transfer probes, a synthetic peptide model system was developed to study stability of the coiled coil (S2 fragment) structure by determining monomer-dimer equilibrium of the peptide. Fluorescence resonance energy transfer and MacroModel software suite were used to obtain distance measurements along with measurement of coiled coil formation. The model peptide was used to characterize the effects of disease-causing-mutations and examine potential candidate drugs (polyamines) to counteract effects of mutations causing HCM. Distance measurements between donor and acceptor probes obtained by computational simulation and fluorescence resonance energy transfer (FRET) were consistent. Measurements also agreed with simulations of unlabeled wildtype, indicating coiled coil structural stability of the peptide. Interaction of the site-specific antibody with the peptide strongly inhibited dimerization and destabilized coiled coil structure of the peptide. Presence of negatively charged glutamate residues in the region of subfragment-2 strongly suggested a potential interaction site for positively charged polyamines. Binding of certain polyamines, such as poly-L-Lysine 11 residues and poly-D-Lysine 17 residues, demonstrated the ability to enhance dimerization and improve stability of the coiled coil structure, while some other polyamines were shown to have insignificant impact on the structure. In an attempt to characterize the effect of HCM-causing-mutations, peptides containing E924K mutation and lethal mutation E930 deletion were synthesized. Fluorescence resonance probes were conjugated to the mutant peptides to determine coiled coil formation. Results obtained from both dynamic simulations and resonance energy transfer experiments indicated that these mutations strongly inhibit dimerization, and thus, destabilize coiled coil structure of the peptide. Further experiments were conducted using heterodimers containing a chain of wildtype and a chain ...
Date: August 2013
Creator: Taei, Nasrin