Spontaneous Rupture and Entanglement of Human Neuronal Tau

Subscriber access provided by Uppsala universitetsbibliotek

Article

Spontaneous Rupture and Entanglement of Human Neuronal Tau Protein Induced by picoNewton Compressive Force S Roy Chowdhury, and H. Peter Lu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00295 • Publication Date (Web): 17 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Spontaneous Rupture and Entanglement of Human Neuronal Tau Protein Induced by picoNewton Compressive Force S. Roy Chowdhury and H. Peter Lu* Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403

Compressive Force Rupture, Human Tau Protein, Atomic Force Microscope, Tau Aggregation, Alzheimer’s disease,

ABSTRACT: Mechanical force vector fluctuations in living cells can have significant impact on protein behavior and functions. Here we report that a human Tau-protein tertiary structure can abruptly and spontaneously rupture, like a balloon, under biologicallyavailable picoNewton compressive force, using a home-modified Atomic Force Microscopy single-molecule manipulation. The rupture behavior is dependent on the physiological level of presence of ions, such as K+ and Mg++. We observed rupture events in the presence of K+ but not in the presence of Mg2+ ions. We have also explored the entangled protein state formed following the events of the multiple and simultaneous protein ruptures under crowding. Crowded proteins simultaneously rupture and then spontaneously refold to an entangled folding state, different from either folded and unfolded states of the Tau protein, can be a plausible pathway for the tau protein aggregation that are related to a number of neuronal degenerated diseases.

INTRODUCTION A growing number of neuronal and pathological evidence suggest that the formation of aggregated tau protein molecules is a pathological hallmark of Alzheimer’s and Parkinson’s diseases. Tau is known to stabilize the microtubule, but in pathological brain tau protein forms neurofibrillary tangles losing its normal function. Elucidating the cause of formation of tau aggregates and loss of their normal functions have been the focus of research to develop better diagnostics and therapeutics.1 Tau is essentially an intrinsically-disordered protein which plays a very important role in stabilizing axonal microtubules in the central nervous system.2 Due to the absence of a fixed tertiary structure, Tau protein is believed to exist as an intrinsically disordered protein in solution. Both circular dichroism measurements3 and electron paramagnetic resonance4, 5 showed a high degree of mobility of the tau protein structure. Tau 441 is an has two inserts at the N-terminal end, a proline-rich region, four repeats (R1, R2, R3, and R4), and a short C-terminal tail.2 Tau mutation can induce tau aggregation which is closely associated with tauopathies.6 For aggregation, the IDP needs to undergo through conformational changes to form the "pro-aggregate". It is observed that Tau repeat domains K18 and K19 can aggregate much faster than the full-length tau.7, 8 Tau mutation in frontotemporal dementia FTDP-17 is known for increasing β-sheet propensity making the structure more prone to aggregation.9-11 Both the molecular basis of early

aggregation events and the mechanism by which tau aggregation causes neuronal dysfunction are still unclear. In a pathogenic form, Tau proteins lose their affinity towards the microtubule, and fold together forming a β-sheet structure12 and aggregates as fibrillary tau.9 Increasing number of evidences suggest that tau protein aggregation is transmittable in neurons in a prion-like manner.13, 14 Tau is a highly water-soluble protein and positively charged in physiological condition. NMR structure study indicates the structural diversity of tau protein in the solution.15 The singlemolecule spectroscopic study has shown evidence that Tau protein is not completely lacking structural motifs. A Forster resonance energy transfer study showed a much less intramolecular distance than a random coil model.16 Single molecular fluorescence polarization anisotropy study showed that tau protein under solution exists in two conformations. These two long-lived conformations adopted by tau protein varies in terms of compactness.17 Atomic force microscopy (AFM) with advanced picoNewton sensitivity and stability is a powerful approach to study protein conformation and fluctuation.18 Pulling force spectroscopy has been studied extensively to explore the details about the dynamics of protein structure and function, and protein-peptide interaction;19-25 whereas compressive force spectroscopy has given more information about rigidity and protein flexibility.26-28 The latter is closely related with protein function under crowding, cell osmotic stress and thermal

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluctuations, domain surface tension, and liquid-liquid phase separation in living cells.29

Page 2 of 13

RESULTS AND DISCUSSIONS Figure 2A shows the typical force curve of the AFM tip engagement with a single Tau protein molecule and its response under an external compressive force. Protein molecules were covalently tethered to a cover glass under buffer solution (pH 7.4). When the AFM tip apex touches the surface of the protein molecule, the compressive force starts loading on the protein. When the force reaches a threshold value, the protein can no longer hold the force and gets spontaneously and suddenly ruptured, which is recorded by a sudden compressive force release (Figure 2A). Figure 2C shows the distribution of rupture force with the rupture force loading distance at 3000 pN/s loading rate.

Figure 1 Cartoon scheme of neurodegeneration process associated with the Tau protein aggregation and detachment from the microtubules of the neurons. The tau protein monomers get separated from the microtubule and forms neurofibrillary tangles (NFTs), destabilizing the microtubule and causing it to degenerate. Previously we have reported compressive force rupture observed in two different globular protein molecules like Calmodulin and HPPK. We have also combined singlemolecule fluorescence resonance energy transfer (smFRET), with conventional AFM technique to get detailed information of the conformational changes in the rupture process in real time with sub-nanometer resolution. The correlated measurement proved that the rupture process is spontaneous and abrupt and free energy driven process.26 We have used this novel behavior to get more information about the protein compactness and flexibility. We found that Calmodulin in its non-activated form shows this compressive fore rupture, whereas in Ca2+-activated form does not go through any rupture event upon compressive force loading.27 Here in this work, we focus our study on exploring the biological impact of such protein ruptures, especially, the multiple proteins rupture simultaneously in crowded environment with a close proximity. Here we report our discovery of the spontaneous tau protein rupture under picoNewton compressive force manipulation and demonstrate both rupture and non-rupture responses from the Tau protein under different electrostatic local environments. We found that tau protein undergoes a spontaneous rupture when the compressive force reaches a picoNewton threshold value depending on the tip approaching speed. Remarkably, the threshold force amplitude is at the thermal fluctuation level available in living cells. We have also found that, in presence Mg2+ ions, tau protein does not show such rupture events, whereas the rupture event retains in presence of monovalent cations like K+. In addition, we have also explored that this kind of spontaneous and simultaneous ruptures of multiple proteins under close proximity can develop entangled protein third state, a entangled state, and can be a plausible mechanism for protein aggregation.

Similar kind of protein rupture under compressive force loading was observed on globular proteins like Calmodulin and HPPK.26, 27 But it is remarkable that an intrinsically disordered protein like tau can also show this behavior. It is already proven that tau can exist in many conformational states, where some are more compact in nature and others are not. This spontaneous protein rupture at the threshold compressive force further proves that protein fluctuation dynamics including dynamic nature of intermolecular hydrogen bonding, inter-domain interactions, friction force with the solvent molecule and inter-domain frictions can induce structural confinement which can withstand pN amount of compressive force applied by the AFM tip apex26, 27, 30. This inherent structural confinement can be closely related to tau protein aggregation.

Figure 2 (A) Typical force curve of a tau protein rupture under compressive force. The sudden drop in the force curve represents the spontaneous rupture of the protein molecule. We have verified this type of spontaneous protein tertiary structure rupture events by systematic control experiments (see Supplementary Figure 4)26, 27 (B) Cartoon scheme of Tau protein rupture under compressive force. (i) The point where there is no physical contact between the AFM tip and the protein molecule. (ii) The molecule gets squeezed under the compressive force after the contact occurs. Black arrows


Page 3 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


represent the force vectors acting on the molecule, AFM tip, and the cover glass. (iii) The compressive force reaches the threshold value, the molecular interaction also got weaken to an amount that it no longer can hold the force, as a result, the protein molecule gets ruptured. (iv) the spontaneously ruptured state of the protein. (C) Distribution of threshold rupture force was plotted against the rupture force loading distance under 3000 pN/s force loading rate. (D) Typical compressive force curve on tau protein under Mg2+ environment. To investigate whether the rupture events are closely related to the local environment, we have found that the protein rupture behaviors are highly sensitive to the charge of the cations in the solution. As we added 2 mM Mg2+ ions we found no rupture event (Figure 2D), which is different from the rupture behavior under K+ environment (88 mM), typical physiological Mg++ and K+ concentrations in living cells31. These different response behaviors under the compressive force are most likely associated with the structural confinement along with the microscopic nature of the internal frictional force and interdomain attraction forces such as hydrogen bonding and electrostatic forces32, 33. An intrinsically disordered protein, like tau, have dynamic properties associated with solvent friction and protein-matrix internal friction34. Both components are dynamic in nature and closely related to the confinement or protein compactness35. The different ionic environment can induce the different amount of confinement to the protein structure and the effective friction along the compressive force rupture coordinate originates from the inter-chain interaction and the solvent interactions around the molecule36. In the presence of K+ environment, these external and internal frictions can enable the protein molecule to hold some compressive force so that a protein structure rupture may occur at the accumulated threshold compressive force; whereas, with the presence of Mg2+ ions, the protein relaxes to a more flexible conformation which does not show any compressive force build-up on the protein as well as any rupture under such force. We propose a comprehensive model for the two types of protein structure responses under compressive force: A protein under compressive force can act either as a “balloon” or a “cotton ball” (see supporting information S1 and Supplementary Figure 2). A balloon may rupture under compressive force and a cotton ball only changes shape but does not rupture. By charge electric field modification, the protein rigidity can be softened to a more flexible form that can diffuse compressive forces by releasing stress to the local environment and through protein shape changes. Here we suggest a possible relation between the electrostatic environment and Tau protein response under compressive force, which can be closely related to protein aggregation and tauopathy37. This kind of behavior is recently reported and existing in a list of globular proteins like HPPK, Calmodulin etc26, 27.

further characterize the rupture threshold force distribution and mean rupture force, we repeated the experiment under different force loading rates (See Supplementary Figure 6). The most probable rupture force was calculated after plotting histograms and fitting it with a Gaussian function. We found that with the increase of the rate of the force loading the rupture force also increases from 25.01 ± 0.87 pN (Figure 3A) to 77.90 ± 0.97 pN, with the force amplitude as low as 5.35 pN. Figure 3B shows the linear dependence of rupture force with the force loading rate. Notably, protein rupture under compressive force is a complex process with a multidimensional force vector matrix. Therefore, a linear fit may only hold in such a small range of loading rate. The actual picture may be different including torsional force distribution inside the protein matrix induced by the AFM tip, multidimensional frictional force, and transient dipole interactions.38 Figure 3D is the calculated loading energy under 3000 pN/s force loading rate. Where the Eloading=0.5*loading distance(l)*Fthreshold .26 Though the loading energy is at 13.97 ± 0.52 kBT, the distribution shows that rupture events can occur at around 10 kBT or lower, which implies that this rupture events can also happen under thermal fluctuation environment in a living cell. Here, kBT at 298K (25C) is 4.11 pNnm or 2.47 kJ/mol. Based on statistical thermodynamics, the Boltzmann equation,39 the population of any molecular states that have energy level bellow 10 kBT is non-negligible or observable at room temperature of 298 K (25 C). For example, if a chemical rate process has an activation barrier at or bellow 10 kBT, there is an observable possibility of such a rate process can occur at room temperature. Using Bell-Evans model (See supporting information) we obtained values of G = 25.98 ± 7.11 kBT (See supporting Information S6). We note that the estimated G value for the free energy difference between the ground state of the folded protein and the protein trasition state at the rupture threshold under the comoressive force may be much more complex. The complexity comes from the local environment including the solvation, tip-protein interaction, and the hydrophobicity domain deformation. Nevertheless, it is intriguing that the protein rupture proceeds as an energetically downhill process, and it is the AFM-applied compressive force to push the protein to the turning point of energetic and structural transition state. The overall free energy change is associated mostly with the sum-over free energy from the protein solvation and protein’s exposing its hydrophobic domains and surfaces to the local environment as the protein deforms and eventually ruptures. It is likely that these free energy changes mostly dominate the free energy changes over the protein internal chain conformational free energy changes to favor the spontaneous protein ruptures.

The protein spontaneous rupture under compressive force is a complex event which includes inhomogeneous local factors like a hydrophilic-hydrophobic force field of the protein molecules and the surrounding. The inhomogeneous nature of the protein rupture gives a broad distribution of the threshold force (Figure 3A). The AFM force loading process is a relatively slow process compared to the protein conformational fluctuation time, which ensures that the rupture process follows an isothermal dynamic with a free-energy downhill kinetics. To



Page 4 of 13

eventually lose its activity and get separated from the microtubule surface forcing it to degenerate.

Figure 3 (A) Protein rupture threshold force distribution under 3000 pN/s loading rates. (B) A linear fit of threshold force with loading rate. (C) Distribution of the rupture force loading distance. (D) Distribution of loading energy under 3000 pN/s force loading rate. To understand the tau protein aggregation it is very important to have the molecular understanding of the earliest step of the protein entanglement process. Simultaneously ruptured proteins in crowding can either refold back to their individual native states or refold into an entanglement state as both pathways are energetically accessible (Figure 4). To investigate the possibility of such entanglement state formation, we have used the conventional force pulling experiment40 by AFM to interrogate the protein structure states after a pair or a number of proteins simultaneously ruptured induced by our compressive force manipulation.

Figure 4 Conceptual presentation of protein entanglement process. Two hands depict two tau protein molecules. A ruptured protein can stay in a metastable state or can refold back to its original state within a few milliseconds or can stay in a different conformational state, based on our experimental observation (see Supporting Information). Under a molecular crowding, such as tau proteins on neuronal microtubules, the compressive force fluctuation can trigger a rare event involving multiple protein ruptures simultaneously, and then simultaneously-ruptured protein molecules can spontaneously fold up into an entangled aggregation state or protein-third-state. It is observed that added tau fibrils extracted from a pathological mice brain to a healthy mice brain induces tau aggregation inside the healthy brain.41 The formation of the protein-third-state may serve as the nucleation of the earlyevent for the Tau protein aggregation and further fibril formation. In the fluctuation of the biological processes, the third-state formation can take the role of bifurcation point at the pathways of forming the Tau fibrils. With such biologically accessible compressive force tau protein molecules can be ruptured under crowding forming the protein-third-state, and

To identify the refolded protein states, we have carried out an AFM pulling experiment followed by compressive force loading induced multiple protein ruptures under crowding. Typically, AFM force pulling curves give clear different features from a refolded single protein vs a multiple protein entangled state42 (see supporting information). We first created ruptured protein molecules under compressive force loading (Figure 5D-i and 4D-ii, and Figure 5E-i and 4E-ii), and then probed the refolded protein states to identify if they are single proteins (Figure 5D-iii and 4D-iv) or refolded-entangled proteins (Figure 5E-iii and 4E-iv) by measuring pulling-rupture force and plotted against the displacement (Figure 5 and Supplementary Figure 7). Figure 5A shows the force-displacement distribution of control experiment where the individual and untangled refolded tau molecules are interrogated by AFM force pulling analysis. Figure 5B shows the force-displacement distribution of the pulling-rupture force of a pair of refolded proteins, after that compressive force ruptures of the paired protein molecules. Interestingly, Figure 5B shows a higher and broader pullingrupture force distribution than that from the control experiment, and more frequent pulling-rupture events recorded in the experiments for paired proteins than that in the control experiment for individual proteins. The results suggest that there is a significant probability of specific interactions that present between a pair of refolded tau proteins when they are both ruptured in a close contact. The higher force values on the pulling curve also indicate the presence of entangled Tau protein formation.42 Figure 5C, subtracting the background data of single Tau proteins (Figure 5A) from the paired Tau proteins (Figure 5B), shows two clearly distinguished regions: The Region I shows the presence of more rupture events which corresponds to lower force and lower displacement in the control experiment, which indicates the unspecific interaction between the tau protein and cover glass surface. Whereas the region II of high force-high displacement indicates the presence of specific interactions in the entangled tau proteins (see supporting information).

Figure 5 2D plot of force vs displacement of pulling-rupture events. (A) A control experiment of individual tau protein pulling force analysis. The experiments are carried out with tau coated AFM tip and trimethoxy silane coated coverglass. Here the observed pulling-rupture forces are due to the interaction


Page 5 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


between the tau protein with the surface, which is unspecific in nature. As a result, the force distribution is in the lower end and also narrow in nature. (B) The pulling force analysis of paired and multiple proteins. The ruptured proteins in the experiment are created by pressing a tau-coated AFM tip on a cover glass surface with attached tau protein molecules to make sure that there are a pair or a multiple of ruptured tau proteins in the pulling force analysis measurement. (C) Subtraction of Figure A from Figure B after normalization. The darker region is specified as region I and the brighter region as region II. Figure C shows the presence of a higher rupture force with longer distance, which indicates the presence of specific interactions between tau proteins or the entangled aggregation. The Bright color separates the signal from the control experiment indicating the presence of specific interactions which is not present in the control experiment. (D) Cartoon scheme of control experiments. (i) The AFM tip approaches the coverglass (ii) The rupture occurs under compressive force loading (iii) AFM tip retraction from the surface and unspecific attraction between the surface and the protein molecule (iv) As the AFM tip moves further, the protein detaches from the surface leaving a pulling-rupture event on the AFM force pulling curve, which is plotted in Figure 5A (E) Cartoon scheme of tau-tau interaction upon compressive force loading. (i) The AFM tip approaches the cover glass with a tau protein on its apex. (ii) A pair of Tau proteins rupture in the close vicinity occurs under compressive force loading, and the ruptured proteins entangle and aggregate while refolding. (iii) AFM tip retraction from the surface and stretches the aggregated protein form (iv) As the AFM tip moves further, the entangled protein molecules disaggregate, leaving single or multiple disentanglement events on the AFM force pulling curve which is plotted in Figure 5B. (iv) A conceptual presentation of multiple protein spontaneously rupture under a compressive force and then simultaneously refolded to a possible entangled states.

DMSO (10% v/v) for 12 hours, and then incubated in 10 nM dimethyl suberimidate in 50 mM PBS (pH 8.0) for 4h. After washing by water and methanol, the glass slide was incubated in 10 nM Tau solutions (PBS, pH 7.4) for 4h. After tethering tau proteins on the cover-glass surface, we take out the coverglass from buffer solution and kept it dry for two days. Then we take the AFM image (Supplementary Figure 8) under HEPES buffer (pH 7.4). The proteins were 1.5-8 nm in heights. Here we note that we did not see tau proteins when we took the AFM image right after the preparation. Our assumption is that with phase change tau protein forms one or multiple conformationally-stable states. Nevertheless, this measurement is essentially for an identification of the tau proteins existence on the coverglass to ensure the sample preparation validity; however, the samples used in our compressive force manipulation are measured under buffer solution. Chemically link Tau protein molecules on the gold coated AFM tip. The Gold coated AFM tip was submerged in 1mM solution of Cysteamine in ethyl alcohol for 6 hours. Then the AFM tip was washed with distilled water and then incubated in 10 nM dimethyl suberimidate in 50 mM PBS (pH 8.0) for 4h. After washing by water and methanol, the AFM tip was incubated in 10 nM Tau solutions (PBS, pH 7.4) for 4h.

ASSOCIATED CONTENT Supporting Information Comparison of Balloon-like form vs the cotton-ball form, AFM force spectroscopy analysis, control experiment, discussion on force mapping experiment, Bell-Evan’s calculation.

AUTHOR INFORMATION Corresponding Author

CONCLUSION

*Corresponding Author: [email protected]

In summary, we have observed abrupt and spontaneous tau protein ruptures under a compressive force ranging from ~5 pN to ~125 pN, at a biologically-available force amplitude range in living cells43. We have also found that this structural response of a protein under compressive force is closely related to the electrostatic local environment. This indicates an imbalance of certain ions can impact the protein structure responses to the compressive force fluctuations, which can trigger the protein entanglements. We have explored multiple-protein rupture-induced entanglements under the introduction of the compressive force in a crowded environment. Our experiment provides an evidence of the entanglement interaction within a pair of refolded tau proteins following a simultaneous and spontaneous paired protein ruptures under crowding. The experimental condition is attainable under the physiological conditions, such as the tau proteins on the neuronal microtubules.

METHODS Chemically link Tau molecules on a cover glass. The cover glass (Gold Seal) was first cleaned and silanized with a mixture of (3-aminopropyl) trimethoxysilane, isobutyltrimethoxysilane with a ratio of 1:1000 dissolved in

Present Addresses Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403

Author Contributions H.P.L. designed research; S.R.C. performed research; H.P.L. contributed new reagents/analytic tools; S.R.C. and H.P.L. analyzed data; and S.R.C. and H.P.L. wrote the paper. Funding Sources Ohio Eminent Scholar Endowment. ACKNOWLEDGMENT We acknowledge the support of this work from the Ohio Eminent Scholar Endowment fund.

REFERENCES 1. Ballatore, C., Lee, V. M.-Y., and Trojanowski, J. Q. (2007) Taumediated neurodegeneration in Alzheimer's disease and related disorders, Nat. Rev. Neurosci. 8, 663. 2. Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E.-M., and Mandelkow, E. (1994) Domains of tau protein and interactions with microtubules, Biochemistry 33, 9511-9522. 3. Akoury, E., Mukrasch, M. D., Biernat, J., Tepper, K., Ozenne, V., Mandelkow, E., Blackledge, M., and Zweckstetter, M. Remodeling of



the conformational ensemble of the repeat domain of tau by an aggregation enhancer, Protein Sci. 25, 1010-1020. 4. Siddiqua, A., and Margittai, M. Three-and Four-repeat Tau Coassemble into Heterogeneous Filaments AN IMPLICATION FOR ALZHEIMER DISEASE, J. Biol. Chem. 285, 37920-37926. 5. Margittai, M., and Langen, R. (2004) Template-assisted filament growth by parallel stacking of tau, Proc. Natl. Acad. Sci. U. S. A. 101, 10278-10283. 6. von Bergen, M., Barghorn, S., Li, L., Marx, A., Biernat, J., Mandelkow, E.-M., and Mandelkow, E. (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local β-structure, J. Biol. Chem. 276, 48165-48174. 7. Ahmed, H. H., Salem, A. M., Atta, H. M., Eskandar, E. F., Farrag, A. R. H., Ghazy, M. A., Salem, N. A., and Aglan, H. A. Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells, World journal of stem cells 8, 106. 8. Barghorn, S., and Mandelkow, E. (2002) Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments, Biochemistry 41, 14885-14896. 9. Schweers, O., Mandelkow, E.-M., Biernat, J., and Mandelkow, E. (1995) Oxidation of cysteine-322 in the repeat domain of microtubuleassociated protein tau controls the in vitro assembly of paired helical filaments, Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467. 10. Gamblin, T. C., King, M. E., Dawson, H., Vitek, M. P., Kuret, J., Berry, R. W., and Binder, L. I. (2000) In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants, Biochemistry 39, 6136-6144. 11. Fodero-Tavoletti, M. T., Okamura, N., Furumoto, S., Mulligan, R. S., Connor, A. R., McLean, C. A., Cao, D., Rigopoulos, A., Cartwright, G. A., and O'keefe, G. 18F-THK523: a novel in vivo tau imaging ligand for Alzheimer's disease, Brain 134, 1089-1100. 12. Von Bergen, M., Barghorn, S., Biernat, J., Mandelkow, E.-M., and Mandelkow, E. (2005) Tau aggregation is driven by a transition from random coil to beta sheet structure, Biochim. Biophys. Acta, Mol. Basis Dis. 1739, 158-166. 13. Brunden, K. R., Trojanowski, J. Q., and Lee, V. M.-Y. (2008) Evidence that non-fibrillar tau causes pathology linked to neurodegeneration and behavioral impairments, J. Alzheimer's Dis. 14, 393-399. 14. Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Clos, A. L., Jackson, G. R., and Kayed, R. Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wildtype mice, Mol. Neurodegener. 6, 39. 15. Mukrasch, M. D., Bibow, S., Korukottu, J., Jeganathan, S., Biernat, J., Griesinger, C., Mandelkow, E., and Zweckstetter, M. (2009) Structural polymorphism of 441-residue tau at single residue resolution, PLoS Biol. 7, e1000034. 16. Jeganathan, S., von Bergen, M., Brutlach, H., Steinhoff, H.-J. r., and Mandelkow, E. (2006) Global hairpin folding of tau in solution, Biochemistry 45, 2283-2293. 17. Manger, L. H., Foote, A. K., Wood, S. L., Holden, M. R., Heylman, K. D., Margittai, M., and Goldsmith, R. H. (2017) Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level, Angew. Chem. 129, 1579015794. 18. Lohr, D., Bash, R., Wang, H., Yodh, J., and Lindsay, S. (2007) Using atomic force microscopy to study chromatin structure and nucleosome remodeling, Methods 41, 333-341. 19. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., and Gaub, H. E. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM, Science 276, 1109-1112. 20. Stigler, J., Ziegler, F., Gieseke, A., Gebhardt, J. C. M., and Rief, M. (2011) The complex folding network of single calmodulin molecules, Science 334, 512-516. 21. Hummer, G., and Szabo, A. (2003) Kinetics from nonequilibrium single-molecule pulling experiments, Biophys. J. 85, 5-15.

Page 6 of 13

22. Onuchic, J. N., Luthey-Schulten, Z., and Wolynes, P. G. (1997) Theory of protein folding: the energy landscape perspective, Annu. Rev. Phys. Chem. 48, 545-600. 23. Paik, D. H., and Perkins, T. T. (2011) Overstretching DNA at 65 pN does not require peeling from free ends or nicks, J. Am. Chem. Soc. 133, 3219-3221. 24. Yang, S., and Cao, J. (2002) Direct measurements of memory effects in single-molecule kinetics, J. Chem. Phys. 117, 10996-11009. 25. Fazal, F. M., and Block, S. M. (2011) Optical tweezers study life under tension, Nat. Photonics 5, 318-321. 26. Chowdhury, S. R., Cao, J., He, Y., and Lu, H. P. (2018) Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation, ACS nano 12, 24482454. 27. Chowdhury, S. R., and Lu, H. P. (2018) Probing Activated and Non-Activated Single Calmodulin Molecules under a Piconewton Compressive Force, Biochemistry 57, 1945-1948. 28. Czajkowsky, D. M., Sun, J., Shen, Y., and Shao, Z. (2015) Single molecule compression reveals intra-protein forces drive cytotoxin pore formation, eLife 4, e08421. 29. Shin, Y., Chang, Y.-C., Lee, D. S. W., Berry, J., Sanders, D. W., Ronceray, P., Wingreen, N. S., Haataja, M., and Brangwynne, C. P. (2018) Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome, Cell 175, 1481-1491. 30. Edwards, S. A., Wagner, J., and Grater, F. Dynamic prestress in a globular protein, PLoS Comput. Biol. 8, e1002509. 31. Milo, R., and Phillips, R. (2016) Cell Biology by the Numbers, 1st ed., Garland Science, New York. 32. Nakamura, H. (1996) Roles of electrostatic interaction in proteins, Q. Rev. Biophys. 29, 1-90. 33. Donhauser, Z. J., Saunders, J. T., D'Urso, D. S., and Garrett, T. A. (2017) Dimerization and Long-Range Repulsion Established by Both Termini of the Microtubule-Associated Protein Tau, Biochemistry 56, 5900-5909. 34. Das, A., and Makarov, D. E. (2018) Dynamics of disordered proteins under confinement: Memory effects and internal friction, J. Phys. Chem. B. 35. Requião, R. D., Fernandes, L., de Souza, H. J. A., Rossetto, S., Domitrovic, T., and Palhano, F. L. (2017) Protein charge distribution in proteomes and its impact on translation, PLoS Comput. Biol. 13, e1005549. 36. Soranno, A., Holla, A., Dingfelder, F., Nettels, D., Makarov, D. E., and Schuler, B. Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations, Proc. Natl. Acad. Sci. U. S. A. 114, E1833-E1839. 37. Gaeta, A., and Hider, R. C. (2005) The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy, Br. J. Pharmacol. 146, 1041-1059. 38. Stacklies, W., Xia, F., and Gräter, F. (2009) Dynamic allostery in the methionine repressor revealed by force distribution analysis, PLoS Comput. Biol. 5, e1000574. 39. Atkins, P., De Paula, J., and Keeler, J. (2018) Atkins' physical chemistry, 11th ed., Oxford university press, New York. 40. Neupane, K., Solanki, A., Sosova, I., Belov, M., and Woodside, M. T. (2014) Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy, PloS one 9, e86495. 41. Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., and Staufenbiel, M. (2009) Transmission and spreading of tauopathy in transgenic mouse brain, Nat. Cell Biol. 11, 909. 42. Barrantes, A., Sotres, J., Hernando-Perez, M., Benitez, M. J., de Pablo, P. J., Baro, A. M., Avila, J., and Jimenez, J. S. (2009) Tau aggregation followed by atomic force microscopy and surface plasmon resonance, and single molecule tau-tau interaction probed by atomic force spectroscopy, J. Alzheimer's Dis. 18, 141-151. 43. Keul, N. D., Oruganty, K., Bergman, E. T. S., Beattie, N. R., McDonald, W. E., Kadirvelraj, R., Gross, M. L., Phillips, R. S., Harvey, S. C., and Wood, Z. A. (2018) The entropic force generated by intrinsically disordered segments tunes protein function, Nature, 1.


Page 7 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 8 of 13

Figure 1


Page 9 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Figure 2



Page 10 of 13

Figure 3


Page 11 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Simultaneous Ruptures

Refolding

Refolding

Compressed

Ruptured

Entangled

Figure 4



Page 12 of 13

Figure 5


Page 13 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Simultaneous Ruptures

Refolding

Refolding

Compressed

Ruptured

Entangled

T.O.C image


Spontaneous Rupture and Entanglement of Human Neuronal Tau

Recommend Documents