Bid 蛋白在誘發細胞凋亡的過程中扮演重要的角色，然而科學家對於其在粒線體外膜上之作用機制以及它的結構穩定性仍未完全瞭解。在此篇論文中，我們將利用生物物理技術去探究這些問題。在第一章中將介紹 BCL-2 家族蛋白質如何調控細胞凋亡，以及 Bid 在其中扮演的角色。在第二章中，我們簡述了此論文主要運用的電子自旋共振光譜 (ESR) 之原理以及其結合蛋白質定點自旋標記 (SDSL) 相關應用。在第三章的研究中，我們探討 Bid 蛋白在粒線體外膜之結構變化以及其作用機制。當細胞進行凋亡時，Bid 蛋白會受到 caspase-8 蛋白酶進行切斷反應形成 cBid，並於粒線體外膜上產生結構變化，導致促凋亡蛋白質 BAX 之活化，最後造成粒線體外膜通透化 (MOMP) 的現象。然而，目前針對Bid蛋白於膜上之結構機制的探討中，普遍使用模仿粒線體外膜組成之脂質環境，而非直接使用粒線體外膜來進行研究。在此章節，我們將萃取出的粒線體與 cBid 在不同條件下反應，並利用 ESR 光譜技術以及定點聚乙二醇修飾方法 (site-directed PEGylation) 去解析 cBid 蛋白在誘發 MOMP 中各階段的結構變化。研究結果顯示 cBid 蛋白在 MOMP 過程中有階段性的結構變化，當 cBid 轉移至粒線體外膜時，會導致其 BH3 helix 暴露但保持整體結構完整性。只有當 cBid 與 BAX 蛋白作用時，cBid 的結構才會劇烈改變，並導致 BAX 蛋白的活化。本研究顯示 cBid 於粒線體上作用機制與文獻中使用仿粒線體脂質環境之結果不同，凸顯粒線體外膜環境對於 cBid 蛋白構形轉變的重要性。第四章中，我們針對 Bid 蛋白的冷變性機制詳細探討。Bid 蛋白於過去研究中被發現具有高熱穩定性，然而與其冷變性相關機制從未有任何研究討論過。本論文研究發現，在 3 M GdnHCl 溶液中，Bid蛋白能夠於290 K附近進行可逆的冷變性反應，且蛋白質結構於 273 K 時完全瓦解。此外，降低溶液中 GdnHCl 之濃度能有效降低Bid 蛋白發生冷變性反應的溫度。藉由 DEER 距離量測以及 ESR 光譜技術，我們進一步解析 Bid 蛋白之三級結構以及局部環境隨著溫度改變造成的變化。本研究顯示，Bid蛋白在 GdnHCl 溶液下處於 marginally stable 狀態，並且主要透過疏水性作用穩定其核心結構。因此，隨著溫度下降，蛋白質之疏水性作用力減弱，造成蛋白質於低溫下結構瓦解，本論文顯示疏水性作用力對於 Bid 蛋白冷變性機制之重要性。

The work presented in this dissertation contains two different topics, whereas both of them are relevant to the BH3 interacting domain death agonist (Bid) protein.
The first topic focuses on revealing the activation mechanism of the pro-apoptotic Bid at mitochondrial membranes. Caspase-8-cleaved Bid (cBid) associates with mitochondria and promotes the activation of BAX, leading to mitochondria outer membrane permeabilization (MOMP) and apoptosis. However, current structural models of cBid are largely based on studies using membrane vesicles and detergent micelles. In this study, we employed spin-label ESR and site-directed PEGylation methods to identify conformations of cBid at real mitochondrial membranes, revealing stepwise mechanisms in the activation process. Upon the binding of cBid to mitochondria, its structure is reorganized to expose the BH3 domain while leaving the structural integrity little altered. The mitochondria-bound cBid remains in the primed state until interacting with BAX. The interaction subsequently triggers the fragmentation of cBid, causes large conformational changes, and promotes BAX-mediated MOMP. Our results reveal structural differences of cBid between mitochondria and other lipid-like environments and, moreover, highlight the role of the membrane binding in modifying cBid structure and assisting the inactive-to-active transition in function.
The second topic investigates the cold denaturation of Bid protein. Bid was previously shown to be a highly thermostable protein, but its cold denaturation has not been reported. We found that in 3 M GdnHCl, Bid can undergo a reversible cold-induced unfolding at temperature around 290 K and the transition temperature decreases with lower concentration of GdnHCl. To explore the mechanism, we employ the QTY (glutamine, threonine, and tyrosine) code approach that makes systematic and specific substitutions of amino acids within the core structure of Bid, allowing us to demonstrate the key role of hydrophobic residues in the protein stability. The conformational changes of Bid before/after the cold denaturation were further confirmed by measuring interspin distances in Bid using the temperature resolved double electron electron resonance (DEER) technique. Cw-ESR spectra of single-labeled Bid were recorded at varying temperature, providing site-specific information concerning the disruption of local structure against temperature. We show that in 3 M GdnHCl, Bid stays in a marginally stable state at room temperatures, primarily stabilized by the hydrophobic residues in the core region. Upon the decrease of temperature, the hydrophobicity of the core residues is decreased, promoting the structure of Bid to be more slack, easily disrupted with lowering temperatures. This study reveals details of how the cold-induced unfolding of a protein can be mediated by hydrophobic interactions.

摘要 i
Abstract ii
謝誌 iv
Table of Contents ix
Abbreviations xiii
CHAPTER 1: Introduction to Bid protein and its role in the mitochondria-mediated apoptosis
1.1 Synopsis 1
1.2 Apoptosis 2
1.3 BCL-2 protein family 4
1.4 BH3-interacting domain death agonist, Bid 9
1.5 Previous studies on the tBid structure on the membrane 11
1.6 Stability study of Bid protein 14
1.7 Motivation of this study 16
CHAPTER 2: Introduction to Electron Spin Resonance
2.1 Principle of Electron Spin Resonance (ESR) 18
2.2 Site-directed spin labeling (SDSL) and nitroxide spectrum 20
2.3 Orientational anisotropy of ESR spectrum 22
2.4 Rotational diffusion of nitroxide spin label and the ESR lineshape 24
2.5 Dipolar interactions between electron spins 26
2.6 Double Electron-Electron resonance (DEER) for distance measurements 30
References for chapter 1 and 2 36
CHAPTER 3: Stepwise activation of the pro-apoptotic protein Bid at mitochondrial membranes
3.1 Introduction 44
3.2 Results 47
3.2.1 Structural integrity of cBid is altered upon the association with mitochondria. 48
3.2.2 The tBid fragment adopts multiple extended conformations at mitochondria. 50
3.2.3 p7 and tBid remain held together in state II, primarily by hydrophobic interactions. 52
3.2.4 PEGylation studies reveal changes in the local environment of individual helices. 53
3.2.5 BH3 domain of α3 is exposed to solvent upon the association with mitochondria. 55
3.2.6 Drastic changes in the local environment of α6–α8 occur during the membrane associations. 55
3.2.7 Membrane-associated cBid interacts with BAX at mitochondria to induce MOMP. 57
3.2.8 No sign of tBid aggregates on mitochondria in state III. 60
3.3 Discussion 60
3.3.1 Conformation of cBid in real mitochondria is unclear from previous studies. 60
3.3.2 The inactive-to-active transition of cBid occurs at mitochondrial membranes. 62
3.4 Conclusions 63
3.5 Materials and Methods 64
3.5.1 Recombinant protein preparation 64
3.5.2 Mitochondria isolation and cytochrome c release assays 67
3.5.3 Cell culture and mitochondria isolation from BAX−/−BAK−/− double knockout HCT116 cells 68
3.5.4 Immunoblotting 69
3.5.5 PEGylation-based gel shift assay 70
3.5.6 Preparation and cw-ESR spectroscopy of spin-labeled cBid 71
3.5.7 DEER distance measurements and molecular modeling 72
3.5.8 Calculation of the number of spins per cluster from DEER data 74
3.6 Supplementary figures 75
References for chapter 3 80
CHAPTER 4: Direct observation of cold denaturation of Bid protein by spin-label ESR
4.1 Introduction 86
4.2 Results 89
4.2.1 Bid undergoes a reversible cold denaturation at temperature above 0 ℃ in the presence of GdnHCl 89
4.2.2 GdnHCl weaken the stability of Bid to promote both heat and cold denaturation 91
4.2.3 Hydrophobic interactions stabilize the structure of Bid in 3 M GdnHCl 93
4.2.4 Study of Bid conformation at various temperature by DEER spectroscopy 96
4.2.5 Cw-ESR reveals local structural changes of Bid due to denaturant and low temperature 98
4.2.6 Spectroscopic evidence for the cold denaturation of Bid 100
4.2.7 Determine changeover temperature during cold denaturation by ESR absorption peak-height analysis 103
4.2.8 Mapping local structural disruptions of Bid during cold denaturation 107
4.3 Discussion 108
4.3.1 Hydrophobic contacts of Bid largely retain at room temperature but become disrupted 
at low temperature in the presence of GdnHCl 109
4.3.2 Cold-induced unfolding events of Bid initiate at similar interface to the heat-induced unfolding 111
4.4 Conclusions 113
4.5 Materials and Methods 114
4.5.1 Bid expression and purification. 114
4.5.2 Circular dichroism (CD) spectroscopy 115
4.5.3 Cw-ESR experiment 117
4.5.4 Peak-height analysis of ESR absorption spectra 117
4.5.5 DEER experiment and analysis 119
4.6 Supplementary figures 121
Reference for chapter 4 136

Chapter 1-2
1. Li, H.L., Zhu, H., Xu, C.J. & Yuan, J.Y. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491-501 (1998).
2. Tait, S.W.G. & Green, D.R. Mitochondria and cell death: outer membrane permeabilization and beyond. Nature Reviews Molecular Cell Biology 11, 621-632 (2010).
3. Kantari, C. & Walczak, H. Caspase-8 and Bid: Caught in the act between death receptors and mitochondria. Biochimica Et Biophysica Acta-Molecular Cell Research 1813, 558-563 (2011).
4. Czabotar, P.E., Lessene, G., Strasser, A. & Adams, J.M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nature Reviews Molecular Cell Biology 15, 49-63 (2014).
5. Singh, R., Letai, A. & Sarosiek, K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nature Reviews Molecular Cell Biology 20, 175-193 (2019).
6. Youle, R.J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology 9, 47-59 (2008).
7. Marino, G., Niso-Santano, M., Baehrecke, E.H. & Kroemer, G. Self-consumption: the interplay of autophagy and apoptosis. Nature Reviews Molecular Cell Biology 15, 81-94 (2014).
8. Strasser, A., Jost, P.J. & Nagata, S. The Many Roles of FAS Receptor Signaling in the Immune System. Immunity 30, 180-192 (2009).
9. Goldstein, J.C., Waterhouse, N.J., Juin, P., Evan, G.I. & Green, D.R. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nature Cell Biology 2, 156-162 (2000).
10. Du, C.Y., Fang, M., Li, Y.C., Li, L. & Wang, X.D. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42 (2000).
11. Verhagen, A.M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43-53 (2000).
12. Moldoveanu, T., Follis, A.V., Kriwacki, R.W. & Green, D.R. Many players in BCL-2 family affairs. Trends in Biochemical Sciences 39, 101-111 (2014).
13. Petros, A.M., Olejniczak, E.T. & Fesik, S.W. Structural biology of the Bcl-2 family of proteins. Biochimica Et Biophysica Acta-Molecular Cell Research 1644, 83-94 (2004).
14. Chipuk, J.E., Moldoveanu, T., Llambi, F., Parsons, M.J. & Green, D.R. The BCL-2 Family Reunion. Molecular Cell 37, 299-310 (2010).
15. Sattler, M. et al. Structure of Bcl-x(L)-Bak peptide complex: Recognition between regulators of apoptosis. Science 275, 983-986 (1997).
16. Petros, A.M. et al. Rationale for Bcl-x(L)/Bad peptide complex formation from structure, mutagenesis, and biophysical studies. Protein Science 9, 2528-2534 (2000).
17. Liu, X.Q., Dai, S.D., Zhu, Y.N., Marrack, P. & Kappler, J.W. The structure of a Bcl-x(L)/Bim fragment complex: implications for bim function. Immunity 19, 341-352 (2003).
18. Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076-U6 (2008).
19. Czabotar, P.E. et al. Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis. Cell 152, 519-531 (2013).
20. Shamas-Din, A., Brahmbhatt, H., Leber, B. & Andrews, D.W. BH3-only proteins: Orchestrators of apoptosis. Biochimica Et Biophysica Acta-Molecular Cell Research 1813, 508-520 (2011).
21. Letai, A. et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183-192 (2002).
22. Walensky, L.D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466-1470 (2004).
23. Oh, K.J. et al. A membrane-targeted BID BCL-2 homology 3 peptide is sufficient for high potency activation of BAX in vitro. Journal of Biological Chemistry 281, 36999-37008 (2006).
24. Certo, M. et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351-365 (2006).
25. Kim, H. et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nature Cell Biology 8, 1348-U19 (2006).
26. Chen, L. et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Molecular Cell 17, 393-403 (2005).
27. Willis, S.N. et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-x(L), but not Bcl-2, until displaced by BH3-only proteins. Genes & Development 19, 1294-1305 (2005).
28. Willis, S.N. et al. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856-859 (2007).
29. Llambi, F. et al. A Unified Model of Mammalian BCL-2 Protein Family Interactions at the Mitochondria. Molecular Cell 44, 517-531 (2011).
30. Wang, K., Yin, X.M., Chao, D.T., Milliman, C.L. & Korsmeyer, S.J. BID: A novel BH3 domain-only death agonist. Genes & Development 10, 2859-2869 (1996).
31. Billen, L.P., Shamas-Din, A. & Andrews, D.W. Bid: a Bax-like BH3 protein. Oncogene 27, S93-S104 (2008).
32. Chou, J.J., Li, H.L., Salvesen, G.S., Yuan, J.Y. & Wagner, G. Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96, 615-624 (1999).
33. Suzuki, M., Youle, R.J. & Tjandra, N. Structure of Bax: Coregulation of dimer formation and intracellular localization. Cell 103, 645-654 (2000).
34. Hinds, M.G. et al. Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death and Differentiation 14, 128-136 (2007).
35. Jhong, S.R. et al. Evidence for an Induced-Fit Process Underlying the Activation of Apoptotic BAX by an Intrinsically Disordered BimBH3 Peptide. Journal of Physical Chemistry B 120, 2751-2760 (2016).
36. Gross, A. et al. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-X-L prevents this release but not tumor necrosis factor-R1/Fas death. Journal of Biological Chemistry 274, 1156-1163 (1999).
37. McDonnell, J.M., Fushman, D., Milliman, C.L., Korsmeyer, S.J. & Cowburn, D. Solution structure of the proapoptotic molecule BID: A structural basis for apoptotic agonists and antagonists. Cell 96, 625-634 (1999).
38. Bleicken, S., Garcia-Saez, A.J., Conte, E. & Bordignon, E. Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria. Plos One 7(2012).
39. Kuwana, T. et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111, 331-342 (2002).
40. Yethon, J.A., Epand, R.F., Leber, B., Epand, R.M. & Andrews, D.W. Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis. Journal of Biological Chemistry 278, 48935-48941 (2003).
41. Huang, K. et al. Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis. Journal of Biological Chemistry 291, 11843-11851 (2016).
42. Lovell, J.F. et al. Membrane Binding by tBid Initiates an Ordered Series of Events Culminating in Membrane Permeabilization by Bax. Cell 135, 1074-1084 (2008).
43. Shamas-Din, A. et al. tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation. Journal of Biological Chemistry 288, 22111-22127 (2013).
44. Korsmeyer, S.J. et al. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death and Differentiation 7, 1166-1173 (2000).
45. Lutter, M. et al. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nature Cell Biology 2, 754-756 (2000).
46. Zha, J.P., Weiler, S., Oh, K.J., Wei, M.C. & Korsmeyer, S.J. Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 1761-1765 (2000).
47. Lutter, M., Perkins, G.A. & Wang, X.D. The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites. Bmc Cell Biology 2(2001).
48. Kim, T.H. et al. Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome c release. Molecular Biology of the Cell 15, 3061-3072 (2004).
49. Kudla, G. et al. The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment. Journal of Biological Chemistry 275, 22713-22718 (2000).
50. Garcia-Saez, A.J., Mingarro, I., Perez-Paya, E. & Salgado, J. Membrane-insertion fragments of Bcl-x(L), Bax, and Bid. Biochemistry 43, 10930-10943 (2004).
51. Gong, X.M. et al. Conformation of membrane-associated proapoptotic tBid. Journal of Biological Chemistry 279, 28954-28960 (2004).
52. Wang, Y. & Tjandra, N. Structural Insights of tBid, the Caspase-8-activated Bid, and Its BH3 Domain. Journal of Biological Chemistry 288, 35840-35851 (2013).
53. Gahl, R.F., Dwivedi, P. & Tjandra, N. Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis. Cell Death & Disease 7(2016).
54. Chan, C.H., Tsai, C.J. & Chiang, Y.W. Side-Chain Packing Interactions Stabilize an Intermediate of BAX Protein against Chemical and Thermal Denaturation. Journal of Physical Chemistry B 119, 54-64 (2015).
55. Hung, C.L., Lin, Y.Y., Chang, H.H. & Chiang, Y.W. Accessing local structural disruption of Bid protein during thermal denaturation by absorption-mode ESR spectroscopy. Rsc Advances 8, 34656-34669 (2018).
56. Rabenstein, M.D. & Shin, Y.K. Determination of the distance between two spin labels attached to a macromolecule. Proceedings of the National Academy of Sciences 92, 8239-8243 (1995).
57. Pake, G.E. Nuclear Resonance Absorption in Hydrated Crystals - Fine Structure of the Proton Line. Journal of Chemical Physics 16, 327-336 (1948).
58. Mchaourab, H.S., Steed, P.R. & Kazmier, K. Toward the Fourth Dimension of Membrane Protein Structure: Insight into Dynamics from Spin-Labeling EPR Spectroscopy. Structure 19, 1549-1561 (2011).
59. Jeschke, G. DEER Distance Measurements on Proteins. Annual Review of Physical Chemistry, Vol 63 63, 419-446 (2012).
60. Georgieva, E.R., Borbat, P.P., Ginter, C., Freed, J.H. & Boudker, O. Conformational ensemble of the sodium-coupled aspartate transporter. Nature Structural & Molecular Biology 20, 215-221 (2013).
61. Sung, T.C. et al. Solution Structure of Apoptotic BAX Oligomer: Oligomerization Likely Precedes Membrane Insertion. Structure 23, 1878-1888 (2015).
62. Tsai, C.J. et al. BAX-Induced Apoptosis Can Be Initiated through a Conformational Selection Mechanism. Structure 23, 139-148 (2015).
63. Dastvan, R., Fischer, A.W., Mishra, S., Meiler, J. & Mchaourab, H.S. Protonation-dependent conformational dynamics of the multidrug transporter EmrE. Proceedings of the National Academy of Sciences of the United States of America 113, 1220-1225 (2016).
64. Schmidt, T., Ghirlando, R., Baber, J. & Clore, G.M. Quantitative Resolution of Monomer-Dimer Populations by Inversion Modulated DEER EPR Spectroscopy. Chemphyschem 17, 2987-2991 (2016).
65. Schmidt, T., Schwieters, C.D. & Clore, G.M. Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR. Proceedings of the National Academy of Sciences of the United States of America 116, 17809-17816 (2019).
66. Chiang, Y.W., Borbat, P.P. & Freed, J.H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. Journal of Magnetic Resonance 172, 279-295 (2005).
67. Chiang, Y.W., Borbat, P.P. & Freed, J.H. Maximum entropy: A complement to Tikhonov regularization for determination of pair distance distributions by pulsed ESR. Journal of Magnetic Resonance 177, 184-196 (2005).

Chapter 3
1. Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59 (2008).
2. Czabotar, P. E., Lessene, G., Strasser, A. & Adams, J. M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell Biol. 15, 49–63 (2014).
3. Tait, S. W. G. & Green, D. R. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11, 621–32 (2010).
4. Li, H., Zhu, H., Xu, C. J. & Yuan, J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501 (1998).
5. Kantari, C. & Walczak, H. Caspase-8 and Bid: Caught in the act between death receptors and mitochondria. Biochimica et Biophysica Acta - Molecular Cell Research vol. 1813 558–563 (2011).
6. Shamas-Din, A., Brahmbhatt, H., Leber, B. & Andrews, D. W. BH3-only proteins: Orchestrators of apoptosis. Biochimica et Biophysica Acta - Molecular Cell Research vol. 1813 508–520 (2011).
7. Leber, B., Geng, F., Kale, J. & Andrews, D. W. Drugs targeting Bcl-2 family members as an emerging strategy in cancer. Expert Rev. Mol. Med. 12, e28 (2010).
8. Hinds, M. G. et al. Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death Differ. 14, 128–36 (2007).
9. Chou, J. J., Li, H., Salvesen, G. S., Yuan, J. & Wagner, G. Solution Structure of BID, an Intracellular Amplifier of Apoptotic Signaling. Cell 96, 615–624 (1999).
10. Petros, A. M., Olejniczak, E. T. & Fesik, S. W. Structural biology of the Bcl-2 family of proteins. Biochim. Biophys. Acta 1644, 83–94 (2004).
11. Suzuki, M., Youle, R. J. & Tjandra, N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103, 645–654 (2000).
12. Kuwana, T. et al. Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane. Cell 111, 331–342 (2002).
13. Yethon, J. A., Epand, R. F., Leber, B., Epand, R. M. & Andrews, D. W. Interaction with a membrane surface triggers a reversible conformational change in Bax normally associated with induction of apoptosis. J. Biol. Chem. 278, 48935–48941 (2003).
14. Huang, K. et al. Cleavage by caspase 8 and mitochondrial membrane association activate the BH3-only protein bid during TRAIL-induced apoptosis. J. Biol. Chem. 291, 11843–11851 (2016).
15. Westphal, D. et al. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane. Proc. Natl. Acad. Sci. U. S. A. 111, E4076–E4085 (2014).
16. Moldoveanu, T. et al. BID-induced structural changes in BAK promote apoptosis. Nat. Struct. Mol. Biol. 20, 589–597 (2013).
17. Lai, Y.-C. et al. The role of cardiolipin in promoting the membrane pore-forming activity of BAX oligomers. Biochim. Biophys. Acta - Biomembr. 1861, 268–280 (2019).
18. Li, M. X. et al. BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove. Proc. Natl. Acad. Sci. 114, 7629–7634 (2017).
19. Gahl, R. F., Dwivedi, P. & Tjandra, N. Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis. Cell Death Dis. 7, e2424 (2016).
20. Wang, Y. & Tjandra, N. Structural insights of tBid, the caspase-8-activated bid, and its BH3domain. J. Biol. Chem. 288, 35840–35851 (2013).
21. Lan, Y.-J., Wang, Y.-T., Hung, C.-L. & Chiang, Y.-W. PEGylation-based strategy to identify pathways involved in the activation of apoptotic BAX protein. Biochim. Biophys. Acta - Gen. Subj. 1864, 129541 (2020).
22. Sung, T.-C. et al. Solution Structure of Apoptotic BAX Oligomer: Oligomerization Likely Precedes Membrane Insertion. Structure 23, 1878–1888 (2015).
23. Jeschke, G. DEER Distance Measurements on Proteins. Annu. Rev. Phys. Chem. 63, 419–446 (2012).
24. Lai, Y., Kuo, Y. & Chiang, Y. Identifying Protein Conformational Dynamics Using Spin-label ESR. Chem. – An Asian J. 14, 3981–3991 (2019).
25. Georgieva, E. R., Borbat, P. P., Ginter, C., Freed, J. H. & Boudker, O. Conformational ensemble of the sodium-coupled aspartate transporter. Nat Struct Mol Biol 20, 215– 221 (2013).
26. Martens, C. et al. Lipids modulate the conformational dynamics of a secondary multidrug transporter. Nat. Struct. Mol. Biol. 23, 744–751 (2016).
27. Kurokawa, T. & Okamura, Y. Mapping of sites facing aqueous environment of voltage- gated proton channel at resting state: A study with PEGylation protection. Biochim. Biophys. Acta - Biomembr. 1838, 382–387 (2014).
28. Robin, A. Y. et al. Ensemble Properties of Bax Determine Its Function. Structure 26, 1346-1359.e5 (2018).
29. McDonald, S. K., Levitz, T. S. & Valiyaveetil, F. I. A Shared Mechanism for the Folding of Voltage-Gated K + Channels. Biochemistry 58, 1660–1671 (2019).
30. Borbat, P. P. & Freed, J. H. Pulse Dipolar Electron Spin Resonance: Distance Measurements. in Structure and Bonding 1–82 (2013). doi:10.1007/430_2012_82.
31. Bode, B. E. et al. Counting the monomers in nanometer-sized oligomers by pulsed electron - Electron double resonance. J. Am. Chem. Soc. 129, 6736–6745 (2007).
32. Chiang, Y.-W., Borbat, P. P. & Freed, J. H. The determination of pair distance
distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–
295 (2005).
33. Chiang, Y.-W., Borbat, P. P. & Freed, J. H. Maximum entropy: A complement to
Tikhonov regularization for determination of pair distance distributions by pulsed
ESR. J. Magn. Reson. 177, 184–196 (2005).
34. Jeschke, G. et al. DeerAnalysis2006—a comprehensive software package for
analyzing pulsed ELDOR data. Appl. Magn. Reson. 30, 473–498 (2006).
35. McDonnell, J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J. & Cowburn, D.
Solution Structure of the Proapoptotic Molecule BID: A Structural Basis for Apoptotic
Agonists and Antagonists. Cell 96, 625–634 (1999).
36. Hagelueken, G., Abdullin, D. & Schiemann, O. mtsslSuite: Probing Biomolecular
Conformation by Spin-Labeling Studies. in Methods in Enzymology 595–622 (2015).
doi:10.1016/bs.mie.2015.06.006.
37. Tina, K. G., Bhadra, R. & Srinivasan, N. PIC: Protein Interactions Calculator. Nucleic
Acids Res. 35, W473-6 (2007).
38. Jubb, H. C. et al. Arpeggio: A Web Server for Calculating and Visualising Interatomic
Interactions in Protein Structures. J. Mol. Biol. 429, 365–371 (2017).
39. Hockings, C. et al. Bid chimeras indicate that most BH3-only proteins can directly
activate Bak and Bax, and show no preference for Bak versus Bax. Cell Death Dis. 6,
e1735 (2015).
40. Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455,
1076–1081 (2008).
41. Czabotar, P. E. et al. Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis. Cell 152, 519–531 (2013).
42. Billen, L. P., Shamas-Din, A. & Andrews, D. W. Bid: a Bax-like BH3 protein. Oncogene 27, S93–S104 (2008).
43. Shamas-Din, A. et al. tBid undergoes multiple conformational changes at the membrane required for bax activation. J. Biol. Chem. 288, 22111–22127 (2013).
44. Shivakumar, S. et al. The proapoptotic protein tbid forms both superficially bound and membrane-inserted oligomers. Biophys. J. 106, 2085–2095 (2014).
45. Oh, K. J. et al. Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study. J. Biol. Chem. 280, 753–67 (2005).
46. Hung, C.-L., Lin, Y.-Y., Chang, H.-H. & Chiang, Y.-W. Accessing local structural disruption of Bid protein during thermal denaturation by absorption-mode ESR spectroscopy. RSC Adv. 8, 34656–34669 (2018).
47. Yamaguchi, R. et al. Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity. Cell Death Differ 14, 616–624 (2007).
48. Wang, C. & Youle, R. J. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak. Oncogene 31, 3177–3189 (2012).
49. Renault, T. T., Floros, K. V & Chipuk, J. E. BAK/BAX activation and cytochrome c release assays using isolated mitochondria. Methods 61, 146–55 (2013).
50. Kelley, L. A., Gardner, S. P. & Sutcliffe, M. J. An automated approach for defining core atoms and domains in an ensemble of NMR-derived protein structures. Protein Eng. Des. Sel. 10, 737–741 (1997).

Chapter 4
1. Czabotar, P.E., Lessene, G., Strasser, A. & Adams, J.M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nature Reviews Molecular Cell Biology 15, 49-63 (2014).
2. Delbridge, A.R.D. & Strasser, A. The BCL-2 protein family, BH3-mimetics and cancer therapy. Cell Death and Differentiation 22, 1071-1080 (2015).
3. Kuwana, T. et al. BH3 domains of BH3-only proteins differentially regulate bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Molecular Cell 17, 525-535 (2005).
4. Youle, R.J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology 9, 47-59 (2008).
5. Shamas-Din, A., Brahmbhatt, H., Leber, B. & Andrews, D.W. BH3-only proteins: Orchestrators of apoptosis. Biochimica Et Biophysica Acta-Molecular Cell Research 1813, 508-520 (2011).
6. Chou, J.J., Li, H.L., Salvesen, G.S., Yuan, J.Y. & Wagner, G. Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96, 615-624 (1999).
7. Bleicken, S., Garcia-Saez, A.J., Conte, E. & Bordignon, E. Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria. Plos One 7(2012).
8. Shamas-Din, A. et al. tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation. Journal of Biological Chemistry 288, 22111-22127 (2013).
9. Wang, Y. & Tjandra, N. Structural Insights of tBid, the Caspase-8-activated Bid, and Its BH3 Domain. Journal of Biological Chemistry 288, 35840-35851 (2013).
10. Gahl, R.F., Dwivedi, P. & Tjandra, N. Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis. Cell Death & Disease 7(2016).
11. Moldoveanu, T. et al. BID-induced structural changes in BAK promote apoptosis. Nature Structural & Molecular Biology 20, 589-+ (2013).
12. Oh, K.J. et al. Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding - A site-directed spin labeling study. Journal of Biological Chemistry 280, 753-767 (2005).
13. Billen, L.P., Shamas-Din, A. & Andrews, D.W. Bid: a Bax-like BH3 protein. Oncogene 27, S93-S104 (2008).
14. Privalov, P.L. Cold Denaturation of Proteins. Critical Reviews in Biochemistry and Molecular Biology 25, 281-305 (1990).
15. Lopez, C.F., Darst, R.K. & Rossky, P.J. Mechanistic elements of protein cold denaturation. Journal of Physical Chemistry B 112, 5961-5967 (2008).
16. Yoshidome, T. & Kinoshita, M. Hydrophobicity at low temperatures and cold denaturation of a protein. Physical Review E 79(2009).
17. Graziano, G. On the mechanism of cold denaturation. Physical Chemistry Chemical Physics 16, 21755-21767 (2014).
18. Pastore, A. et al. Unbiased cold denaturation: Low- and high-temperature unfolding of yeast frataxin under physiological conditions. Journal of the American Chemical Society 129, 5374-+ (2007).
19. Baez, M., Wilson, C.A.M., Ramirez-Sarmiento, C.A., Guixe, V. & Babul, J. Expanded Monomeric Intermediate upon Cold and Heat Unfolding of Phosphofructokinase-2 from Escherichia coli. Biophysical Journal 103, 2187-2194 (2012).
20. Buchner, G.S., Shih, N., Reece, A.E., Niebling, S. & Kubelka, J. Unusual Cold Denaturation of a Small Protein Domain. Biochemistry 51, 6496-6498 (2012).
21. Jaremko, M. et al. Cold denaturation of a protein dimer monitored at atomic resolution. Nature Chemical Biology 9, 264-270 (2013).
22. Yan, R., DeLos Rios, P., Pastore, A. & Temussi, P.A. The cold denaturation of IscU highlights structure-function dualism in marginally stable proteins. Communications Chemistry 1(2018).
23. Agashe, V.R. & Udgaonkar, J.B. Thermodynamics of Denaturation of Barstar - Evidence for Cold Denaturation and Evaluation of the Interaction with Guanidine-Hydrochloride. Biochemistry 34, 3286-3299 (1995).
24. Huang, G.W.S. & Oas, T.G. Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent. Biochemistry 35, 6173-6180 (1996).
25. Shan, B., McClendon, S., Rospigliosi, C., Eliezer, D. & Raleighr, D.P. The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure. Journal of the American Chemical Society 132, 4669-4677 (2010).
26. Vajpai, N., Nisius, L., Wiktor, M. & Grzesiek, S. High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin. Proceedings of the National Academy of Sciences of the United States of America 110, E368-E376 (2013).
27. Babu, C.R., Hilser, V.J. & Wand, A.J. Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation. Nature Structural & Molecular Biology 11, 352-357 (2004).
28. Van Horn, W.D., Simorellis, A.K. & Flynn, P.F. Low-temperature studies of encapsulated proteins. Journal of the American Chemical Society 127, 13553-13560 (2005).
29. Pometun, M.S., Peterson, R.W., Babu, C.R. & Wand, A.J. Cold denaturation of encapsulated ubiquitin. Journal of the American Chemical Society 128, 10652-10653 (2006).
30. Whitten, S.T., Kurtz, A.J., Pometun, M.S., Wand, A.J. & Hilser, V.J. Revealing the nature of the native state ensemble through cold denaturation. Biochemistry 45, 10163-10174 (2006).
31. Zhang, S.G. et al. QTY code enables design of detergent-free chemokine receptors that retain ligand-binding activities. Proceedings of the National Academy of Sciences of the United States of America 115, E8652-E8659 (2018).
32. Qing, R. et al. QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity. Proceedings of the National Academy of Sciences of the United States of America 116, 25668-25676 (2019).
33. Klug, C.S., Su, W.Y., Liu, J., Klebba, P.E. & Feix, J.B. Denaturant Unfolding of the Ferric Enterobactin Receptor and Ligand-Induced Stabilization Studied by Site-Directed Spin-Labeling. Biochemistry 34, 14230-14236 (1995).
34. Lindgren, M. et al. Characterization of a Folding Intermediate of Human Carbonic-Anhydrase-Ii - Probing Local Mobility by Electron-Paramagnetic-Resonance. Biophysical Journal 69, 202-213 (1995).
35. Svensson, M. et al. Mapping the Folding Intermediate of Human Carbonic-Anhydrase .2. Probing Substructure by Chemical-Reactivity and Spin and Fluorescence Labeling of Engineered Cysteine Residues. Biochemistry 34, 8606-8620 (1995).
36. Jeschke, G. DEER Distance Measurements on Proteins. Annual Review of Physical Chemistry, Vol 63 63, 419-446 (2012).
37. Sung, T.C. et al. Solution Structure of Apoptotic BAX Oligomer: Oligomerization Likely Precedes Membrane Insertion. Structure 23, 1878-1888 (2015).
38. Tsai, C.J. et al. BAX-Induced Apoptosis Can Be Initiated through a Conformational Selection Mechanism. Structure 23, 139-148 (2015).
39. Lai, Y.C., Kuo, Y.H. & Chiang, Y.W. Identifying Protein Conformational Dynamics Using Spin-label ESR. Chemistry-an Asian Journal 14, 3981-3991 (2019).
40. Hung, C.L., Lin, Y.Y., Chang, H.H. & Chiang, Y.W. Accessing local structural disruption of Bid protein during thermal denaturation by absorption-mode ESR spectroscopy. Rsc Advances 8, 34656-34669 (2018).
41. Becktel, W.J. & Schellman, J.A. Protein Stability Curves. Biopolymers 26, 1859-1877 (1987).
42. Szyperski, T., Mills, J.L., Perl, D. & Balbach, J. Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of Delta C-p of protein unfolding. European Biophysics Journal with Biophysics Letters 35, 363-366 (2006).
43. Pastore, A., Martin, S.R. & Temussi, P.A. Generalized View of Protein Folding: In Medio Stat Virtus. Journal of the American Chemical Society 141, 2194-2200 (2019).
44. McDonnell, J.M., Fushman, D., Milliman, C.L., Korsmeyer, S.J. & Cowburn, D. Solution structure of the proapoptotic molecule BID: A structural basis for apoptotic agonists and antagonists. Cell 96, 625-634 (1999).
45. Kao, T.Y., Tsai, C.J., Lan, Y.J. & Chiang, Y.W. The role of conformational heterogeneity in regulating the apoptotic activity of BAX protein. Physical Chemistry Chemical Physics 19, 9584-9591 (2017).
46. Chiang, Y.W., Borbat, P.P. & Freed, J.H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. Journal of Magnetic Resonance 172, 279-295 (2005).
47. Chiang, Y.W., Borbat, P.P. & Freed, J.H. Maximum entropy: A complement to Tikhonov regularization for determination of pair distance distributions by pulsed ESR. Journal of Magnetic Resonance 177, 184-196 (2005).
48. Hagelueken, G., Abdullin, D. & Schiemann, O. mtsslSuite: Probing Biomolecular Conformation by Spin-Labeling Studies. Electron Paramagnetic Resonance Investigations of Biological Systems by Using Spin Labels, Spin Probes, and Intrinsic Metal Ions, Pt A 563, 595-622 (2015).
49. Tang, X.L. & Pikal, M.J. The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying. Pharmaceutical Research 22, 1167-1175 (2005).
50. Street, T.O., Bolen, D.W. & Rose, G.D. A molecular mechanism for osmolyte-induced protein stability. Proceedings of the National Academy of Sciences of the United States of America 103, 13997-14002 (2006).
51. Dubey, V. & Daschakraborty, S. Influence of glycerol on the cooling effect of pair hydrophobicity in water: relevance to proteins' stabilization at low temperature. Physical Chemistry Chemical Physics 21, 800-812 (2019).
52. Skalicky, J.J., Sukumaran, D.K., Mills, J.L. & Szyperski, T. Toward structural biology in supercooled water. Journal of the American Chemical Society 122, 3230-3231 (2000).
53. Szyperski, T. & Mills, J.L. NMR-based structural biology of proteins in supercooled water. Journal of Structural and Functional Genomics 12, 1 (2011).
54. Mason, P.E., Brady, J.W., Neilson, G.W. & Dempsey, C.E. The interaction of guanidinium ions with a model peptide. Biophysical journal 93, L04-L6 (2007).
55. O'Brien, E.P., Dima, R.I., Brooks, B. & Thirumalai, D. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: Lessons for protein denaturation mechanism. Journal of the American Chemical Society 129, 7346-7353 (2007).
56. Dempsey, C.E., Mason, P.E. & Jungwirth, P. Complex Ion Effects on Polypeptide Conformational Stability: Chloride and Sulfate Salts of Guanidinium and Tetrapropylammonium. Journal of the American Chemical Society 133, 7300-7303 (2011).
57. Huerta-Viga, A. & Woutersen, S. Protein Denaturation with Guanidinium: A 2D-IR Study. Journal of Physical Chemistry Letters 4, 3397-3401 (2013).
58. Scott, J.N., Nucci, N.V. & Vanderkooi, J.M. Changes in Water Structure Induced by the Guanidinium Cation and Implications for Protein Denaturation. Journal of Physical Chemistry A 112, 10939-10948 (2008).
59. Pazos, I.M. & Gai, F. Solute's Perspective on How Trimethylamine Oxide, Urea, and Guanidine Hydrochloride Affect Water's Hydrogen Bonding Ability. Journal of Physical Chemistry B 116, 12473-12478 (2012).
60. Cooper, R.J., Heiles, S., DiTucci, M.J. & Williams, E.R. Hydration of Guanidinium: Second Shell Formation at Small Cluster Size. Journal of Physical Chemistry A 118, 5657-5666 (2014).
61. Jha, S.K. & Marqusee, S. Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride. Proceedings of the National Academy of Sciences of the United States of America 111, 4856-4861 (2014).
62. Mason, P.E., Neilson, G.W., Dempsey, C.E., Barnes, A.C. & Cruickshank, J.M. The hydration structure of guanidinium and thiocyanate ions: Implications for protein stability in aqueous solution. Proceedings of the National Academy of Sciences of the United States of America 100, 4557-4561 (2003).
63. van der Post, S.T., Tielrooij, K.J., Hunger, J., Backus, E.H.G. & Bakker, H.J. Femtosecond study of the effects of ions and hydrophobes on the dynamics of water. Faraday Discussions 160, 171-189 (2013).
64. Mason, P.E., Dempsey, C.E., Neilson, G.W., Kline, S.R. & Brady, J.W. Preferential Interactions of Guanidinum Ions with Aromatic Groups over Aliphatic Groups. Journal of the American Chemical Society 131, 16689-16696 (2009).
65. Mason, P.E. et al. Specificity of Ion-Protein Interactions: Complementary and Competitive Effects of Tetrapropylammonium, Guanidinium, Sulfate, and Chloride Ions. Journal of Physical Chemistry B 113, 3227-3234 (2009).
66. Monera, O.D., Kay, C.M. & Hodges, R.S. Protein Denaturation with Guanidine-Hydrochloride or Urea Provides a Different Estimate of Stability Depending on the Contributions of Electrostatic Interactions. Protein Science 3, 1984-1991 (1994).
67. Camilloni, C. et al. Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations. Biophysical Journal 94, 4654-4661 (2008).
68. Meuzelaar, H., Panman, M.R. & Woutersen, S. Guanidinium-Induced Denaturation by Breaking of Salt Bridges. Angewandte Chemie-International Edition 54, 15255-15259 (2015).
69. Vondrasek, J., Mason, P.E., Heyda, J., Collins, K.D. & Jungwirth, P. The Molecular Origin of Like-Charge Arginine-Arginine Pairing in Water. Journal of Physical Chemistry B 113, 9041-9045 (2009).
70. Kubickova, A. et al. Guanidinium Cations Pair with Positively Charged Arginine Side Chains in Water. Journal of Physical Chemistry Letters 2, 1387-1389 (2011).
71. Tina, K.G., Bhadra, R. & Srinivasan, N. PIC: Protein Interactions Calculator. Nucleic Acids Research 35, W473-W476 (2007).
72. Jubb, H.C. et al. Arpeggio: A Web Server for Calculating and Visualising Interatomic Interactions in Protein Structures. Journal of Molecular Biology 429, 365-371 (2017).
73. Dias, C.L., Ala-Nissila, T., Karttunen, M., Vattulainen, I. & Grant, M. Microscopic mechanism for cold denaturation. Physical Review Letters 100(2008).
74. Dias, C.L. et al. The hydrophobic effect and its role in cold denaturation. Cryobiology 60, 91-99 (2010).
75. Wolfenden, R., Lewis, C.A., Yuan, Y. & Carter, C.W. Temperature dependence of amino acid hydrophobicities. Proceedings of the National Academy of Sciences of the United States of America 112, 7484-7488 (2015).
76. van Dijk, E., Hoogeveen, A. & Abeln, S. The Hydrophobic Temperature Dependence of Amino Acids Directly Calculated from Protein Structures. Plos Computational Biology 11(2015).
77. van Dijk, E., Varilly, P., Knowles, T.P.J., Frenkel, D. & Abeln, S. Consistent Treatment of Hydrophobicity in Protein Lattice Models Accounts for Cold Denaturation. Physical Review Letters 116(2016).
78. Pucci, F. & Rooman, M. Physical and molecular bases of protein thermal stability and cold adaptation. Current Opinion in Structural Biology 42, 117-128 (2017).
79. Wang, K., Yin, X.M., Chao, D.T., Milliman, C.L. & Korsmeyer, S.J. BID: A novel BH3 domain-only death agonist. Genes & Development 10, 2859-2869 (1996).
80. Desagher, S. et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. Journal of Cell Biology 144, 891-901 (1999).
81. Sarig, R. et al. BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. Journal of Biological Chemistry 278, 10707-10715 (2003).
82. Valentijn, A.J. & Gilmore, A.P. Translocation of full-length bid to mitochondria during anoikis. Journal of Biological Chemistry 279, 32848-32857 (2004).
83. Grigoryants, V.M., DeWeerd, K.A. & Scholes, C.P. Method of rapid mix EPR applied to the folding of bi-spin-labeled protein as a probe for the dynamic onset of interaction between sequentially distant side chains. Journal of Physical Chemistry B 108, 9463-9468 (2004).
84. Chiang, Y.W., Shimoyama, Y., Feigenson, G.W. & Freed, J.H. Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance. Biophysical Journal 87, 2483-2496 (2004).
85. Fischler, M.A. & Bolles, R.C. Random Sample Consensus - a Paradigm for Model-Fitting with Applications to Image-Analysis and Automated Cartography. Communications of the Acm 24, 381-395 (1981).
86. Georgieva, E.R. et al. Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme. Journal of Magnetic Resonance 216, 69-77 (2012).