1. Molecular insights into the reversible formation of tau protein fibrils Yin Luo, a Paul Dinkel, b Xiang Yu, c Martin Margittai, b Jie Zheng, c Ruth Nussinov, d,e Guanghong Wei, a and Buyong Ma d a State Key Laboratory of Surface Physics, Department of Physics, Fudan University, Shanghai, P. R. China. E-mail: ghwei@fudan.edu.cnghwei@fudan.edu.cn b Department of Chemistry & Biochemistry, University of Denver, Denver, Colorado 80208, USA. E-mail: martin.margittai@du.edumartin.margittai@du.edu c Department of Chemical & Biomolecular Engineering, The University of Akron, Akron, Ohio 44325, USA. E-mail: zhengj@uakron.eduzhengj@uakron.edu d Basic science Program, SAIC-Frederick, Inc. CCRNP, Frederick National Lab, Frederick, Maryland 21702, USA. E-mail: mabuyong@mail.nih.govmabuyong@mail.nih.gov e Sackler Inst. of Molecular Medicine. Tel Aviv University, Tel Aviv 69978, Israel. E-mail: ruthnu@helix.nih.govruthnu@helix.nih.gov Introduction Functional amyloids can be used in biofilms and in engineering nanomaterials, while pathological amyloids can cause neurodegenerative disorders. The aggregation of the microtubule-associated tau protein into neurofibrillary tangles is considered as one of the important factors leading to Alzheimer disease. Tau proteins may have four or three microtubule binding repeats, with both repeat forms found in the fibrils of the brain of Alzheimer’s patients. The shortened tau proteins containing only the four repeats (K18) and three repeats (K19) have been widely used in studies of tau protein aggregation, since the remaining regions only form a disordered fuzzy coat. Using a combined computational and experimental approach, we first studied the monomeric and oligomeric structures of K18 (130 aa) and K19 (99 aa), and then examined the stability of fibril-like oligomers. We found that the formation of tau protein fibril displays temperature-dependent feature and the fibril formed at elevated temperature can dissociated into monomer upon cooling. Models and Methods Computational Approach  Method: replica exchange molecular dynamics (REMD)  GROMACS 4.5.3 using Charmm27 force field (explicit water and ions)  Monomer system (48 replicas, 310 ~ 430 K)  Octamer system (64 replicas, 310 ~ 380 K) Experimental Approach  Aggregation of K18 and K19 in vitro with or without heparin at 275 K and 343 K  Electron paramagnetic resonance (EPR)  Transmission electron microscope (TEM) Initial Conformations--monomer  21 homology modeling structures (5 of 21 listed below as the first five structures)  3 structures built with U-turn like structures (1 of 1 listed below at the last) Initial Conformations--octamer  32 fibril-like octamers (8 different packing model) 2, 3  32 random octameric structures References 1.Luo, Y., Dinkel, P., Yu, X., Margittai, M., Zheng, J., Nussinov, R., Wei, G., Ma, B. Chem. Commun. 2013, 49, 3582-3584. 2.Siddiqua, A., Luo, Y., Meyer, V., Swanson, M. A., Yu, X., Wei, G., Zheng, J., Eaton, G. R., Ma, B., Nussinov, R., Eaton, S. S., Margittai, M. J. Am. Chem. Soc. 2012, 134, 10271-10278. 3.Yu, X., Luo, Y., Dinkel, P., Zheng, J., Wei, G., Margittai, M., Nussinov, R., Ma, B. J. Biol. Chem. 2012, 287, 14950-14959. 4.Mukrasch, D. M., Biernat, J., von Bergen, M., Griesinger, C., Mandelkow, E., Zweckstetter, M. J. Biol. Chem. 2005, 280, 24978-24986. 5.Ozenne, V., Schneider, R., Yao, M., Huang, J. R., Salmon, L., Zwechkstetter, M., Jensen, R. M., and Blackledge, M. J. Am. Chem. Soc. 2012, 134, 15138-15148. 6.Kim, H. Y., Cho, M. K., Riedel, D., Fernandez, O. C, and Zweckstetter, M. Angew. Chem. Int. Ed. 2008, 47, 5046-5048. Conclusions 1.The formation of tau fibrils at 343 K can be reversed at around 275 K. 2.Heparin locks the tau fibrils and prevents its reversion. 3.The temperature-dependent stabilization of the fibril is coupled with its temperature-sensitive monomeric properties, driven by both hydrophobic and electrostatic interactions. 4.Temperature dependent aggregation for intrinsically disordered proteins possibly are characterized by a high content of hydrophilic and charged residues. + + = Solvation energy = + Protein-Protein interaction energy Simulation results of octamer systems Temperature-dependent  -sheet propensities Results Simulation results of monomer systems Predicted by SPARTA program Highly correlated with the NMR experiment data. 4 A.Although tau protein is an intrinsically disordered protein, there are still some regions with high  -sheet propensities (residues 275-280, 298-311 and 330- 340), close to those obtained by NMR conformation sampling. 5 B.The temperature-dependent solvent accessible surface area (SASA). C.The temperature-dependent  - probability. The b-sheet propensities increase as the temperature increases. Favorable hydrophobic and electrostatic interactions toward fibril formation at higher temperature. Solvation energies are dominated by the protein- solvent interaction. The less compact random conformations adopted at lower temperature are attributed to the electrostatic part of protein- solvent interactions. Experimental observation (K18) bar=200 nm A.Fast aggregation with heparin (within minutes), slow without heparin (within hours). B.Fibril formed in the absence of heparin appeared to disintegrate when mounted onto the grids. C.With heparin, amyloid fibrils formed at 343 K kept stable after cooling to 275 K, with the main peak of acrylodan fluorescence bule- shifted a litter further. D.Without heparin, amyloid fibrils formed at 343 K disappeared after cooling to 275 K, with the main peak of acrylodan fluorescence red shifted back to the original monomeric position.