臺灣博碩士論文加值系統

TDP-43是一個在生物中廣泛存在的蛋白質，在DNA/RNA的調控上扮演著重要的角色。它由414個胺基酸所組成，包含了N端，兩個RNA辨識區域，以及一段沒有特定三級結構的C端。在病理學上，TDP-43是肌萎縮性脊髓側索硬化症中，病理性蛋白質堆積的最主要成分，不過其詳細的病理機轉仍不清楚。而近幾年來，除了蛋白質的沉澱以外，科學家還發現了另一個可能的致病機轉，稱為蛋白質的相變。蛋白質的相變指的是有些蛋白質會因為自身或環境因素的改變，而聚集成如微滴的形式，這樣的轉變是可回復的，與一般的蛋白質沉澱有所不同；而TDP-43也有這樣的現象。科學家認為，相變的平衡對蛋白質的恆定是十分重要的，平衡的破壞也有可能導致疾病的產生。以前的研究已證實，不管是關於蛋白質沉澱或相變，TDP-43的C端都是最關鍵的區段，且幾乎所有的疾病突變都位於C端上，這些都表示TDP-43的C端非常的重要。不過，由於TDP-43的C端非常容易沉澱，使得相關的研究仍十分有限。在本篇研究中，為了探討可能的致病機制，我們討論了TDP-43 C端wild type與三個疾病上發現的突變，利用大腸桿菌表現並純化後，使用NMR光譜儀與其它生物物理的技術，對TDP-43 C端的二級結構、動力學，以及相變的性質上做系統性的分析。我們發現TDP-43 C端中有一區段有a-helix的結構傾向，且它可以藉由自我聚集，使得蛋白質有更多的機會進行相變，我們也發現相變的發生是疏水性作用力所吸引，靜電作用力所排斥的平衡；而病理上的突變並不會影響蛋白質結構，也僅稍微改變了一點自我聚集的傾向，但確實會影響相變的傾向。這些發現有助於我們對TDP-43 C端沉澱與相變的分子機制有更深入的了解。

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a DNA/RNA binding protein which contains an N-terminal domain, two RNA-recognition motifs (RRM1 and RRM2), and a glycine-rich C-terminal domain. TDP-43 is the critical protein of amyotrophic laterals sclerosis (ALS). The proteinopathy and liquid-liquid phase separation (LLPS) are related to TDP-43’s intrinsically disordered C-terminal domain (TDP-43C), but the mechanisms remain elusive. To understand the mechanism of the aggregation and LLPS for TDP-43C, it is critical to obtain the information of protein structure and dynamic at residue-specific level. There are some major challenges for studying TDP-43C. TDP-43C is an aggregation-prone protein which is difficult to purify and usually affected by the purification protocol or storage conditions. We optimized the standard purification protocol which with careful protein quality control. We observed the TDP-43C could process LLPS and form the droplet phase which was turbid at low temperature. This phenomenon is reversible at high temperature. We used NMR spectroscopy and other optical methods to systematically analyze the protein structure, protein dynamics and the property of LLPS of TDP-43C wild type and disease-related mutations. We demonstrated TDP-43C had a-helix secondary structure in the middle part and the disease-related mutations did not significantly affect the secondary structure propensity. TDP-43C processes self-association through the -helix region and some mutations slightly affect the self-association equilibrium. The self-association is mediated by hydrophobic and electrostatic force, and we found the tryptophan (residue 334) is critical for phase separation.

誌謝......i
摘要......ii
Abstract......iii
Contents......iv
List of figures......vi
Chapter 1 Introduction......1
1.1 Physiological function of TDP-43......1
1.2 Pathological mechanism of TDP-43......2
1.3 Domains of TDP-43......3
1.4 Phase separation of TDP-43......8
1.5 Aim......9
Chapter 2 Materials and methods......10
2.1 Materials......10
2.1.1 Table of material......10
2.1.2 Table of primer......13
2.1.3 Table of buffer......14
2.2 Methods......17
2.2.1 DNA construct and primer design......17
2.2.2 Protein expression and purification......18
2.2.3 Circular dichroism spectroscopy......21
2.2.4 Nuclear Magnetic Resonance spectroscopy......23
2.2.5 Analytical ultracentrifuge......26
2.2.6 Turbidity assay......27
Chapter 3 Results......28
3.1 The protein quality control is important for TDP-43 C-terminus......28
3.2 The disease-associated mutations do not significantly affect the secondary structure propensity of TDP-43 C-terminus......30
3.3 The intensity profiles indicate the difference of protein kinetics......34
3.4 The changes of signal intensities of a-helix region indicate self-association......37
3.5 The disease mutants, TDP-43CM337V and TDP-43CQ331K, slightly reduce the population and propensity of self-association......41
3.6 The LLPS forms are undetectable on NMR and CD......44
3.7 The mutations show the different LLPS propensity, and the LLPS is a balance of hydrophobic and electrostatic forces......46
3.8 The tryptophan in the -helix region is critical for phase separation......49
3.9 TDP-43 C-terminus shows the equilibrium of different states with two separate energy barriers......51
Chapter 4 Discussion......52
4.1 TDP-43 C-terminal domain has the dynamic a-helical secondary structure in the central region......52
4.2 The disease mutations slightly reduce the self-association, but this is not major reason to the abolition of phase separation......52
4.3 TDP-43 C-terminal domain undergoes phase separation at low temperature......54
4.4 Phase separation of TDP-43 is a precise balance in the neurodegenerative diseases......55
Reference......57
Appendix......65

Figure 1. The structure of TDP-43 and distribution of disease mutation......7
Figure 2. The schematic illustration of phase separation......8
Figure 3. The major constructs in the experiments......17
Figure 4. Schematic illustration of circular dichroism spectroscopy and the examples of different secondary structure......22
Figure 5. Schematic illustration of analytical ultracentrifugation......26
Figure 6. Purification and quality control of TDP-43 C-terminus......29
Figure 7. The HSQC spectra of TDP-43 C-terminus......31
Figure 8. The NMR secondary structure analysis of TDP-43 C-terminus......32
Figure 9. The CD secondary structure analysis of TDP-43 C-terminus......33
Figure 10. The intensity profiles of TDP-43 C-terminus......35
Figure 11. Schematic illustration of the features of intensities profiles......36
Figure 12. The α-helical structure at different temperatures......38
Figure 13. The conformation exchange model on NMR spectrum......39
Figure 14. The self-association model of TDP-43 C-terminus......40
Figure 15. Comparison of the self-association of TDP-43 C-terminus of difference constants......43
Figure 16. The NMR intensity with LLPS forms......44
Figure 17. The CD spectra with LLPS forms......45
Figure 18. The analysis of NMR spectra of wild type at low temperature (with phase separation)......46
Figure 19. The properties of phase separation of TDP-43 C-terminus......48
Figure 20. The tryptophan of residue 334 is important for phase separation......50
Figure 21. Summary of TDP-43C structure, self-association and phase separation......56

1. Wu, L.S., et al., TDP-43, a neuro-pathosignature factor, is essential for early mouse embryogenesis. Genesis, 2010. 48(1): p. 56-62.
2. Lee, E.B., V.M. Lee, and J.Q. Trojanowski, Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration. Nat Rev Neurosci, 2011. 13(1): p. 38-50.
3. Sephton, C.F., et al., Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem, 2011. 286(2): p. 1204-15.
4. Tollervey, J.R., et al., Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci, 2011. 14(4): p. 452-8.
5. Buratti, E., et al., TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing. J Biol Chem, 2005. 280(45): p. 37572-84.
6. Freibaum, B.D., et al., Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. J Proteome Res, 2010. 9(2): p. 1104-20.
7. Buratti, E., et al., Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J, 2001. 20(7): p. 1774-84.
8. Mohagheghi, F., et al., TDP-43 functions within a network of hnRNP proteins to inhibit the production of a truncated human SORT1 receptor. Hum Mol Genet, 2016. 25(3): p. 534-45.
9. De Conti, L., et al., TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways. Nucleic Acids Res, 2015. 43(18): p. 8990-9005.
10. Colombrita, C., et al., From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models. Biochim Biophys Acta, 2015. 1849(12): p. 1398-410.
11. Ling, S.C., et al., ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A, 2010. 107(30): p. 13318-23.
12. Ayala, Y.M., T. Misteli, and F.E. Baralle, TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression. Proc Natl Acad Sci U S A, 2008. 105(10): p. 3785-9.
13. Polymenidou, M., et al., Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci, 2011. 14(4): p. 459-68.
14. Godena, V.K., et al., TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization. PLoS One, 2011. 6(3): p. e17808.
15. Romano, M., F. Feiguin, and E. Buratti, TBPH/TDP-43 modulates translation of Drosophila futsch mRNA through an UG-rich sequence within its 5'UTR. Brain Res, 2016. 1647: p. 50-6.
16. Ayala, Y.M., et al., Human, Drosophila, and C.elegans TDP43: nucleic acid binding properties and splicing regulatory function. J Mol Biol, 2005. 348(3): p. 575-88.
17. Fan, Z., X. Chen, and R. Chen, Transcriptome-wide analysis of TDP-43 binding small RNAs identifies miR-NID1 (miR-8485), a novel miRNA that represses NRXN1 expression. Genomics, 2014. 103(1): p. 76-82.
18. King, I.N., et al., The RNA-binding protein TDP-43 selectively disrupts microRNA-1/206 incorporation into the RNA-induced silencing complex. J Biol Chem, 2014. 289(20): p. 14263-71.
19. Zhang, Z., et al., Downregulation of microRNA-9 in iPSC-derived neurons of FTD/ALS patients with TDP-43 mutations. PLoS One, 2013. 8(10): p. e76055.
20. Liu, X., et al., Long non-coding RNA gadd7 interacts with TDP-43 and regulates Cdk6 mRNA decay. EMBO J, 2012. 31(23): p. 4415-27.
21. Guo, F., et al., Regulation of MALAT1 expression by TDP43 controls the migration and invasion of non-small cell lung cancer cells in vitro. Biochem Biophys Res Commun, 2015. 465(2): p. 293-8.
22. Higashi, S., et al., TDP-43 associates with stalled ribosomes and contributes to cell survival during cellular stress. J Neurochem, 2013. 126(2): p. 288-300.
23. Elvira, G., et al., Characterization of an RNA granule from developing brain. Mol Cell Proteomics, 2006. 5(4): p. 635-51.
24. Wang, I.F., et al., TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor. J Neurochem, 2008. 105(3): p. 797-806.
25. Neumann, M., et al., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science, 2006. 314(5796): p. 130-3.
26. Deng, H.X., et al., Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature, 2011. 477(7363): p. 211-5.
27. Zhang, Y.J., et al., Phosphorylation regulates proteasomal-mediated degradation and solubility of TAR DNA binding protein-43 C-terminal fragments. Mol Neurodegener, 2010. 5: p. 33.
28. Liachko, N.F., C.R. Guthrie, and B.C. Kraemer, Phosphorylation promotes neurotoxicity in a Caenorhabditis elegans model of TDP-43 proteinopathy. J Neurosci, 2010. 30(48): p. 16208-19.
29. Cohen, T.J., et al., An acetylation switch controls TDP-43 function and aggregation propensity. Nat Commun, 2015. 6: p. 5845.
30. Igaz, L.M., et al., Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies. J Biol Chem, 2009. 284(13): p. 8516-24.
31. Nonaka, T., et al., Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43. Hum Mol Genet, 2009. 18(18): p. 3353-64.
32. Zhang, Y.J., et al., Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci U S A, 2009. 106(18): p. 7607-12.
33. Igaz, L.M., et al., Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest, 2011. 121(2): p. 726-38.
34. Qin, H., et al., TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA. Proc Natl Acad Sci U S A, 2014. 111(52): p. 18619-24.
35. Mompean, M., et al., The TDP-43 N-terminal domain structure at high resolution. FEBS J, 2016. 283(7): p. 1242-60.
36. Johnson, B.S., et al., A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity. Proc Natl Acad Sci U S A, 2008. 105(17): p. 6439-44.
37. Li, H.Y., et al., Hyperphosphorylation as a defense mechanism to reduce TDP-43 aggregation. PLoS One, 2011. 6(8): p. e23075.
38. Bozzo, F., et al., Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells. Neurobiol Dis, 2016. 94: p. 63-72.
39. Chang, C.K., et al., The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity. Biochem Biophys Res Commun, 2012. 425(2): p. 219-24.
40. Zhang, Y.J., et al., The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation. Hum Mol Genet, 2013. 22(15): p. 3112-22.
41. Winton, M.J., et al., A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett, 2008. 582(15): p. 2252-6.
42. Kuo, P.H., et al., The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids. Nucleic Acids Res, 2014. 42(7): p. 4712-22.
43. Kuo, P.H., et al., Structural insights into TDP-43 in nucleic-acid binding and domain interactions. Nucleic Acids Res, 2009. 37(6): p. 1799-808.
44. Lukavsky, P.J., et al., Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Nat Struct Mol Biol, 2013. 20(12): p. 1443-9.
45. Chiang, C.H., et al., Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation. Sci Rep, 2016. 6: p. 21581.
46. Huang, Y.C., et al., Inhibition of TDP-43 aggregation by nucleic acid binding. PLoS One, 2013. 8(5): p. e64002.
47. Moreno, F., et al., A novel mutation P112H in the TARDBP gene associated with frontotemporal lobar degeneration without motor neuron disease and abundant neuritic amyloid plaques. Acta Neuropathol Commun, 2015. 3: p. 19.
48. Furukawa, Y., et al., A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43. Sci Rep, 2016. 6: p. 20576.
49. Schnabel, J., Protein folding: The dark side of proteins. Nature, 2010. 464(7290): p. 828-9.
50. Chouard, T., Structural biology: Breaking the protein rules. Nature, 2011. 471(7337): p. 151-3.
51. Chen, T.C., et al., The Nearest-Neighbor Effect on Random-Coil NMR Chemical Shifts Demonstrated Using a Low-Complexity Amino-Acid Sequence. Protein Pept Lett, 2016. 23(11): p. 967-975.
52. Johnson, B.S., et al., TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity. J Biol Chem, 2009. 284(30): p. 20329-39.
53. Capitini, C., et al., TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells. PLoS One, 2014. 9(1): p. e86720.
54. Jiang, L.L., et al., Structural transformation of the amyloidogenic core region of TDP-43 protein initiates its aggregation and cytoplasmic inclusion. J Biol Chem, 2013. 288(27): p. 19614-24.
55. Mompean, M., et al., Structural Evidence of Amyloid Fibril Formation in the Putative Aggregation Domain of TDP-43. J Phys Chem Lett, 2015. 6(13): p. 2608-15.
56. Wang, I.F., et al., The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies. Nat Commun, 2012. 3: p. 766.
57. Budini, M., et al., Cellular model of TAR DNA-binding protein 43 (TDP-43) aggregation based on its C-terminal Gln/Asn-rich region. J Biol Chem, 2012. 287(10): p. 7512-25.
58. Walker, A.K., et al., ALS-associated TDP-43 induces endoplasmic reticulum stress, which drives cytoplasmic TDP-43 accumulation and stress granule formation. PLoS One, 2013. 8(11): p. e81170.
59. Estes, P.S., et al., Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis. Dis Model Mech, 2013. 6(3): p. 721-33.
60. Sun, C.S., et al., The influence of pathological mutations and proline substitutions in TDP-43 glycine-rich peptides on its amyloid properties and cellular toxicity. PLoS One, 2014. 9(8): p. e103644.
61. Zhu, L., et al., An ALS-mutant TDP-43 neurotoxic peptide adopts an anti-parallel beta-structure and induces TDP-43 redistribution. Hum Mol Genet, 2014. 23(25): p. 6863-77.
62. Gopal, P.P., et al., Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons. Proc Natl Acad Sci U S A, 2017. 114(12): p. E2466-E2475.
63. Ihara, R., et al., RNA binding mediates neurotoxicity in the transgenic Drosophila model of TDP-43 proteinopathy. Hum Mol Genet, 2013. 22(22): p. 4474-84.
64. Braun, R.J., et al., Neurotoxic 43-kDa TAR DNA-binding protein (TDP-43) triggers mitochondrion-dependent programmed cell death in yeast. J Biol Chem, 2011. 286(22): p. 19958-72.
65. Choksi, D.K., et al., TDP-43 Phosphorylation by casein kinase Iepsilon promotes oligomerization and enhances toxicity in vivo. Hum Mol Genet, 2014. 23(4): p. 1025-35.
66. Dong, H., et al., Curcumin abolishes mutant TDP-43 induced excitability in a motoneuron-like cellular model of ALS. Neuroscience, 2014. 272: p. 141-53.
67. Li, Z., et al., The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila. Hum Mol Genet, 2013. 22(2): p. 218-25.
68. Mitchell, J.C., et al., Wild type human TDP-43 potentiates ALS-linked mutant TDP-43 driven progressive motor and cortical neuron degeneration with pathological features of ALS. Acta Neuropathol Commun, 2015. 3: p. 36.
69. Janssens, J., et al., Overexpression of ALS-associated p.M337V human TDP-43 in mice worsens disease features compared to wild-type human TDP-43 mice. Mol Neurobiol, 2013. 48(1): p. 22-35.
70. Liu, Y.C., P.M. Chiang, and K.J. Tsai, Disease animal models of TDP-43 proteinopathy and their pre-clinical applications. Int J Mol Sci, 2013. 14(10): p. 20079-111.
71. Xu, Y.F., et al., Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice. Mol Neurodegener, 2011. 6: p. 73.
72. Alami, N.H., et al., Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron, 2014. 81(3): p. 536-43.
73. Radivojac, P., et al., Prediction of boundaries between intrinsically ordered and disordered protein regions. Pac Symp Biocomput, 2003: p. 216-27.
74. Obradovic, Z., et al., Exploiting heterogeneous sequence properties improves prediction of protein disorder. Proteins, 2005. 61 Suppl 7: p. 176-82.
75. Dosztanyi, Z., et al., IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics, 2005. 21(16): p. 3433-4.
76. Toretsky, J.A. and P.E. Wright, Assemblages: functional units formed by cellular phase separation. J Cell Biol, 2014. 206(5): p. 579-88.
77. Molliex, A., et al., Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell, 2015. 163(1): p. 123-33.
78. Lin, Y., et al., Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. Mol Cell, 2015. 60(2): p. 208-19.
79. Patel, A., et al., A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell, 2015. 162(5): p. 1066-77.
80. Burke, K.A., et al., Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II. Mol Cell, 2015. 60(2): p. 231-41.
81. Schmidt, H.B. and R. Rohatgi, In Vivo Formation of Vacuolated Multi-phase Compartments Lacking Membranes. Cell Rep, 2016. 16(5): p. 1228-36.
82. Conicella, A.E., et al., ALS Mutations Disrupt Phase Separation Mediated by alpha-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure, 2016. 24(9): p. 1537-49.
83. Frey, S., R.P. Richter, and D. Gorlich, FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science, 2006. 314(5800): p. 815-7.
84. Xiang, S., et al., The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei. Cell, 2015. 163(4): p. 829-39.
85. Cioce, M. and A.I. Lamond, Cajal bodies: a long history of discovery. Annu Rev Cell Dev Biol, 2005. 21: p. 105-31.
86. Lallemand-Breitenbach, V. and H. de The, PML nuclear bodies. Cold Spring Harb Perspect Biol, 2010. 2(5): p. a000661.
87. Buchan, J.R., mRNP granules. Assembly, function, and connections with disease. RNA Biol, 2014. 11(8): p. 1019-30.
88. Greenfield, N.J., Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc, 2006. 1(6): p. 2876-90.
89. Sreerama, N. and R.W. Woody, Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem, 2000. 287(2): p. 252-60.
90. Whitmore, L. and B.A. Wallace, DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res, 2004. 32(Web Server issue): p. W668-73.
91. Piotto, M., V. Saudek, and V. Sklenar, Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR, 1992. 2(6): p. 661-5.
92. Hyberts, S.G., et al., Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. J Biomol NMR, 2012. 52(4): p. 315-27.
93. Delaglio, F., et al., NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR, 1995. 6(3): p. 277-93.
94. Goddard, T.D. and D.G. Kneller, Sparky 3. San Francisco, University of California. 2005.
95. Hyberts, S.G., et al., FM reconstruction of non-uniformly sampled protein NMR data at higher dimensions and optimization by distillation. J Biomol NMR, 2009. 45(3): p. 283-94.
96. Chou, C.Y., Y.H. Hsieh, and G.G. Chang, Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses. Methods, 2011. 54(1): p. 76-82.
97. Borcherds, W., et al., Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells. Nat Chem Biol, 2014. 10(12): p. 1000-2.
98. Sun, Y. and A. Chakrabartty, Phase to Phase with TDP-43. Biochemistry, 2017. 56(6): p. 809-823.
99. Brangwynne, C.P., P. Tompa, and R.V. Pappu, Polymer physics of intracellular phase transitions. NATURE PHYSICS, 2015. 11,: p. 899-904.
100. Lim, L., et al., ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43. PLoS Biol, 2016. 14(1): p. e1002338.
101. Mompean, M., et al., Electrostatic Repulsion Governs TDP-43 C-terminal Domain Aggregation. PLoS Biol, 2016. 14(4): p. e1002447.
102. Nott, T.J., et al., Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol Cell, 2015. 57(5): p. 936-47.
103. Zhang, H., et al., RNA Controls PolyQ Protein Phase Transitions. Mol Cell, 2015. 60(2): p. 220-30.
104. Beck, M., et al., The quantitative proteome of a human cell line. Mol Syst Biol, 2011. 7: p. 549.
105. Wang, W., et al., The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity. Nat Med, 2016. 22(8): p. 869-78.