This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are developing a new technique for the analysis of protein aggregation by monitoring the extent to which specific protein sites are exposed to water. This approach relies on the principle of dynamic nuclear polarization (DNP) of solutions, also known as the Overhauser effect, where the coupling between electron and proton is mediated by translational molecular motion of solution state molecules. By saturating one of the electron spin resonances (ESR) of unpaired electrons of stable nitroxide radicals we are able to transfer part of its polarization to a coupled proton resulting in an increased NMR signal. Enhanced signal of water protons in combination with site-specific spin labeling of proteins gives information about the local solvent dynamics at specific protein sites. We were able to utilize such characterization in studying aggregation of tau proteins because local solvent dynamics changes dramatically where protein sites undergo binding and aggregation. The aggregation kinetics was monitored in this fashion at various sites of the tau protein. Our DNP-derived kinetic data was interpreted and verified by several other established methods such as EM, fluorescence and turbidity measurements. Observation of broadening of ESR signal over time was also employed to obtain kinetic data of tau aggregation based on modulation of inter-tau proximity between spin labels and/or change of rotational dynamics of the different tau species (monomer, aggregates etc.). Kinetic rates of tau aggregation were observed due to varying concentration of inducer molecules for aggregation, here heparin. Decrease in aggregation rates was noted with change in average chain length of heparin from 3 to 6 to 18 kDa. Kinetic data from several spin-labeled mutants were compared, in which most mutants reveal elongated fiber morphology by EM that is typical for amyloid plaques, while one mutant does not form fibers but amorphous agglomerates. We would like to obtain images of the tau fibers as they form in an aggregation process under ambient solution condition in situ, to accompany and support our characterization of tau aggregation in the solution state studied by dynamic nuclear polarization technique. We are the first group to develop a DNP based approach to monitor protein aggregation processes in solution state. Therefore, the Cryo TEM images of tau fibers in a frozen solution state will not represent key experimental data for our study, but will present an important control experiment and would serve as a supplemental material for our experiments. We would like to directly observe the morphology and size of the fibers at various time points of aggregation from 10 minutes after the start of aggregation to up to 24 hours of the fiber formation process.
| Ebeida, Mohamed S; Rushdi, Ahmad A; Awad, Muhammad A et al. (2016) Disk Density Tuning of a Maximal Random Packing. Comput Graph Forum 35:259-269 |
| Wensel, Theodore G; Zhang, Zhixian; Anastassov, Ivan A et al. (2016) Structural and molecular bases of rod photoreceptor morphogenesis and disease. Prog Retin Eye Res 55:32-51 |
| Bucero, Marta Abril; Bajaj, Chandrajit; Mourrain, Bernard (2016) On the construction of general cubature formula by flat extensions. Linear Algebra Appl 502:104-125 |
| Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I (2015) Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment. Eur J Transl Myol 25:4803 |
| Rushdi, Ahmad A; Mitchell, Scott A; Bajaj, Chandrajit L et al. (2015) Robust All-quad Meshing of Domains with Connected Regions. Procedia Eng 124:96-108 |
| Edwards, John; Daniel, Eric; Pascucci, Valerio et al. (2015) Approximating the Generalized Voronoi Diagram of Closely Spaced Objects. Comput Graph Forum 34:299-309 |
| Wensel, Theodore G; Gilliam, Jared C (2015) Three-dimensional architecture of murine rod cilium revealed by cryo-EM. Methods Mol Biol 1271:267-92 |
| Jeter, Cameron B; Patel, Saumil S; Morris, Jeffrey S et al. (2015) Oculomotor executive function abnormalities with increased tic severity in Tourette syndrome. J Child Psychol Psychiatry 56:193-202 |
| Zhang, Qin; Cha, Deukhyun; Bajaj, Chandrajit (2015) Quality Partitioned Meshing of Multi-Material Objects. Procedia Eng 124:187-199 |
| Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I (2015) Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment. Eur J Transl Myol 25:35-48 |
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