結語

在此論文中，我們證實了利用 over-critical folding process 能夠將蛋白質 PGB1 摺疊至其自然態，並且摺疊後的蛋白質擁有與一般生理環境的功能相同的功能。

藉由收集 over-critical folding process 各個摺疊階段的摺疊中間態，我們觀察到了 PGB1 的摺疊過程為多階層反應，與許多 文獻研究的推論相符合並 且給予很直接 的證據支持此論點。另外，以色胺酸和 IAEDANS 很適合做為用來觀察蛋白質內 部動態變化的 FRET 供體和受體。

參考文獻

1. Bruce Alberts AJ, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter (2007) The Shape of the Protein. Molecular Biology of the Cell, (Garland Science), 5 Ed.

2. Anfinsen CB (1973) Principles that govern the folding of protein chains.

Science 181:223-230.

3. Misawa S & Kumagai I (1999) Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies. Peptide Science 51:297-307.

4. Chang C-C, Su Y-C, Cheng M-S, & Kan L-S (2002) Protein folding by a quasi-static-like process: A first-order state transition. Physical Review E 66:021903.

5. Chang CC, Tsai CT, & Chang CY (2002) Structural restoration of inactive recombinant fish growth hormones by chemical chaperonin and solvent restraint approaches. Protein engineering 15:437.

6. Liu Y-L, Lee H-T, Chang C-C, & Kan L-S (2003) Reversible folding of cysteine-rich metallothionein by an overcritical reaction path. Biochemical and Biophysical Research Communications 306:59-63.

7. Chang C-C, Yeh X-C, Lee H-T, Lin P-Y, & Kan L-S (2004) Refolding of lysozyme by quasistatic and direct dilution reaction paths: A first -order-like state transition. Physical Review E 70:011904.

8. Ptitsyn OB (1994) Kinetic and equilibrium intermediates in protein folding.

Protein engineering 7:593-596.

9. Agashe VR, Shastry MCR, & Udgaonkar JB (1995) Initial hydrophobic collapse in the folding of barstar. Nature 377:754-757.

10. Abkevich VI, Gutin AM, & Shakhnovich EI (1994) Specific Nucleus as the Transition State for Protein Folding: Evidence from the Lattice Model.

Biochemistry 33:10026-10036.

11. Wetlaufer DB (1973) Nucleation, Rapid Folding, and Globular Intrachain Regions in Proteins. Proceedings of the National Academy of Sciences 70:697-701.

12. Guo Z & Thirumalai D (1995) Kinetics of protein folding: Nucleation mechanism, time scales, and pathways. Biopolymers 36:83-102.

collision model and experimental data. Protein Science 3:650-668.

14. Karplus M & Weaver DL (1979) Diffusion–collision model for protein folding.

Biopolymers 18:1421-1437.

15. Bjorck L & Kronvall G (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology 133:969-974.

16. Akerström B & Björck L (1986) A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. Journal of Biological Chemistry 261:10240-10247.

17. Fahnestock SR, Alexander P, Nagle J, & Filpula D (1986) Gene for an immunoglobulin-binding protein from a group G streptococcus. J. Bacteriol.

167:870-880.

18. Sjöbring U, Björck L, & Kastern W (1991) Streptococcal protein G. Gene structure and protein binding properties. Journal of Biological Chemistry 266:399-405.

19. Sjobring U, Trojnar J, Grubb A, Akerstrom B, & Bjorck L (1989) Ig-binding bacterial proteins also bind proteinase inhibitors. The Journal of Immunology 143:2948-2954.

20. Gronenborn AM & Clore GM (1993) Identification of the contact surface of a streptococcal protein G domain complexed with a human Fc fragme nt. Journal of molecular biology 233:331-335.

21. Sauer-Eriksson AE, Kleywegt GJ, Uhlen M, & Jones TA (1995) Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure 3:265-278.

22. Bjorck L & Kronvall G (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology 133:969.

23. Akerstrom B, Brodin T, Reis K, & Bjorck L (1985) Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. The Journal of Immunology 135:2589.

24. Gronenborn AM, Filpula DR, Essig NZ, Achari A, Whitlow M, Wingfield PT,

& Clore G (1991) A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253:657.

25. Achari A, Hale SP, Howard AJ, Clore GM, Gronenborn AM, Hardman KD, &

Whitlow M (1992) 1.67 Å X-ray structure of the B2 immunoglobulin-binding

domain of streptococcal protein G and comparison to the NMR structure of the B1 domain. Biochemistry 31:10449-10457.

26. Alexander P, Fahnestock S, Lee T, Orban J, & Bryan P (1992) Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

Biochemistry 31:3597-3603.

27. Blanco FJ, Rivas G, & Serrano L (1994) A short linear peptide that folds into a native stable β-hairpin in aqueous solution. Nature structural & molecular biology 1:584-590.

28. Munoz V, Thompson PA, Hofrichter J, & Eaton WA (1997) Folding dynamics and mechanism of β-hairpin formation. Nature 390:196-198.

29. Jackson SE (1998) How do small single-domain proteins fold? Folding and Design 3:R81-R91.

30. Park S, Shastry M, & Roder H (1999) Folding dynamics of the B1 dom ain of protein G explored by ultrarapid mixing. nature structural biology 6:943-947.

31. Krantz B, Mayne L, Rumbley J, Englander S, & Sosnick T (2002) Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding. Journal of molecular biology 324:359-371.

32. Roder H, Maki K, & Cheng H (2006) Early Events in Protein Folding Explored by Rapid Mixing Methods. Chemical Reviews 106:1836-1861.

33. Kuszewski J, Clore GM, & Gronenborn AM (1994) Fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein G.

Protein Science: A Publication of the Protein Society 3:1945.

34. Frank MK, Clore GM, & Gronenborn AM (1995) Structural and dynamic characterization of the urea denatured state of the immunog lobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy. Protein Science: A Publication of the Protein Society 4:2605.

35. Park S, O'Neil K, & Roder H (1997) An Early Intermediate in the Folding Reaction of the B1 Domain of Protein G Contains a Native-like Core.

Biochemistry 36:14277-14283.

36. Kmiecik S & Kolinski A (2008) Folding pathway of the B1 domain of protein G explored by multiscale modeling. Biophysical journal 94:726-736.

37. Kolinski A (2004) Protein modeling and structure prediction with a reduced representation. ACTA BIOCHIMICA POLONICA-ENGLISH EDITION-

38. McCallister EL, Alm E, & Baker D (2000) Critical role of [beta] -hairpin formation in protein G folding. Nat Struct Mol Biol 7:669-673.

39. Lacowicz J (2006) Principles of fluorescence spectroscopy (Springer) 3rd Ed.

40. Eftink MR & Ghiron CA (1976) Fluorescence quenching of indole and model micelle systems. The Journal of Physical Chemistry 80:486-493.

41. Eftink MR & Ghiron CA (1976) Exposure of tryptophanyl residues in proteins.

Quantitative determination by fluorescence quenching studies. Biochemistry 15:672-680.

42. Eftink MR & Ghiron CA (1977) Exposure of tryptophanyl residues and protein dynamics. Biochemistry 16:5546-5551.

43. Grabar KC, Freeman RG, Hommer MB, & Natan MJ (1995) Preparation and Characterization of Au Colloid Monolayers. Analytical Chemistry 67:735-743.

44. Daniel M-C & Astruc D (2003) Gold Nanoparticles:  Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chemical Reviews 104:293-346.

45. Liang X, Wang Z, & Liu C (2010) Size-Controlled Synthesis of Colloidal Gold Nanoparticles at Room Temperature Under the Influence of Glow Discharge . Nanoscale research letters 5:124-129.

46. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz.

Annalen der Physik 437:55-75.

47. Pollok BA & Heim R (1999) Using GFP in FRET-based applications. Trends in cell biology 9:57-60.

48. Jares-Erijman EA & Jovin TM (2003) FRET imaging. Nature Biotechnology 21:1387-1395.

49. Sekar RB & Periasamy A (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. The Journal of cell biology 160:629.

50. Kenworthy AK, Petranova N, & Edidin M (2000) High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Molecular Biology of the Cell 11:1645.

51. Jha S & Udgaonkar J (2009) Direct evidence for a dry molten g lobule intermediate during the unfolding of a small protein. Proceedings of the National Academy of Sciences 106:12289.

52. Thakur A, Jayaraman M, Mishra R, Thakur M, Chellgren V, Byeon I, Anjum D,

Kodali R, Creamer T, & Conway J (2009) Polyglutamine disru ption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism.

Nature structural & molecular biology 16:380-389.

53. Hudson EN & Weber G (1973) Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry 12:4154-4161.

54. Hermanson GT (1996) Bioconjugate techniques (Academic Pr).

55. Bartlett JMS & Stirling D (2003) PCR protocols (Humana Pr Inc).

56. Sambrook J & Russell DW (2001) Molecular cloning: a laboratory manual (Cold spring harbor laboratory press).

57. Davis B (1964) Disc electrophoresis. 11. Method and application to human serum proteins. Ann. NY Acad. Sci 121:404¡V427.

58. Shapiro AL, Viñuela E, & V. Maizel J (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS -polyacrylamide gels.

Biochemical and Biophysical Research Communications 28:815-820.

59. Weber K & Osborn M (1969) The Reliability of Molecular Weight Determinations by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.

Journal of Biological Chemistry 244:4406-4412.

60. Mirsky AE & Pauling L (1936) On the structure of native, denatured, and coagulated proteins. Proceedings of the National Academy of Sciences of the United States of America 22:439.

61. Welch WJ & Brown CR (1996) Influence of molecular and chemical chaperones on protein folding. Cell stress & chaperones 1:109.

62. Kasha M (1952) Collisional Perturbation of Spin Orbital Coupling and the Mechanism of Fluorescence Quenching. A Visual Demonstration of the Perturbation. The Journal of chemical physics 20:71.

63. Altekar W (1974) The quenching of the tryptophyl and tyrosyl fluorescence of proteins by cesium ion. FEBS Letters 49:208-211.

64. Vos R & Engelborghs* Y (1994) A FLUORESCENCE STUDY OF TRYPTOPHAN-HISTIDINE INTERACTIONS IN THE PEPTIDE ANANTIN AND IN SOLUTION. Photochemistry and Photobiology 60:24-32.

65. Li HY, Ng EKO, Lee SMY, Kotaka M, Tsui SKW, Lee CY, Fung KP, & Waye MMY (2001) Protein¡Vprotein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two fusion fluorescence resonance energy transfer (FRET). Journal of cellular

66. Ambasta RK, Kumar P, Griendling KK, Schmidt HHHW, Busse R, & Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. Journal of Biological Chemistry 279:45935.

67. Hillisch A, Lorenz M, & Diekmann S (2001) Recent advances in FRET:

distance determination in protein-DNA complexes. Current Opinion in Structural Biology 11:201-207.

68. Takaoka A, Wang ZC, Choi MK, Yanai H, Negishi H, Ban T, Lu Y, Miyagishi M, Kodama T, & Honda K (2007) DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448:501-505.

69. Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, & Ebright RH (2004) Catabolite activator protein: DNA binding and transcription activa tion.

Current Opinion in Structural Biology 14:10-20.

70. Ray PC, Fortner A, & Darbha GK (2006) Gold Nanoparticle Based FRET Asssay for the Detection of DNA Cleavage. The Journal of Physical Chemistry B 110:20745-20748.

71. Millar DP (1996) Fluorescence st udies of DNA and RNA structure and dynamics. Current Opinion in Structural Biology 6:322-326.

72. Huang F, Settanni G, & Fersht A (2008) Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain. Protein Engineering Design and Selection.

73. Magg C & Schmid FX (2004) Rapid Collapse Precedes the Fast Two -state Folding of the Cold Shock Protein. Journal of molecular biology 335:1309-1323.

74. Miyake-Stoner S, Miller A, Hammill J, Peeler J, Hess K, Mehl R, & Brewer S (2009) Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan. Biochemistry 48:5953-5962.

75. Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182:319-326.

76. Johnson Jr WC (1988) Secondary structure of proteins through circular dichroism spectroscopy. Annual review of biophysics and biophysical chemistry 17:145-166.

77. Kelly SM & Price NC (1997) The application of circular dichroism to studies of protein folding and unfolding. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1338:161-185.

78. Deléage G & Geourjon C (1993) An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Computer applications in the biosciences : CABIOS 9:197-199.

79. Sreerama N & Woody RW (1993) A Self-Consistent Method for the Analysis of Protein Secondary Structure from Circular Dichroism. Analytical Biochemistry 209:32-44.

80. Sreerama N, Venyaminov SYU, & Woody RW (1999) Estimation of the number of α-helical and β-strand segments in proteins using circular dichroism spectroscopy. Protein Science 8:370-380.

81. Shirley BA (1995) Urea and guanidine hydrochloride denaturation curves.

Methods Mol Biol 40:177-190.

82. Tcherkasskaya O, Knutson J, Bowley S, Frank M, & Gronenborn A (2000) Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1. Biochemistry 39:11216-11226.

83. Frishman D & Argos P (1995) Knowledge-based protein secondary structure assignment. Proteins-Structure Function and Genetics 23:566-579.

84. Carson M, Johnson DH, McDonald H, Brouillette C, & DeLucas LJ (2007) His-tag impact on structure. Acta Crystallographica Section D: Biological Crystallography 63:295-301.

85. TEALE FW (1960) The ultraviolet fluorescence of proteins in neutral solution.

Biochem. J. 76:381-380.

86. Burstein EA, Vedenkina NS, & Ivkova MN (1973) Fluorescence and the Location of Tryptophan Residues in Protein Molecules. Photochemistry and Photobiology 18:263-279.

87. Muin o PL & Callis PR (2008) Solvent Effects on the Fluorescence Quenching of Tryptophan by Amides via Electron Transfer. Experimental and Computational Studies. The Journal of Physical Chemistry B 113:2572-2577.

88. Callis PR & Burgess BK (1997) Tryptophan Fluorescence Shifts in Proteins from Hybrid Simulations:  An Electrostatic Approach. The Journal of Physical Chemistry B 101:9429-9432.

89. Pan C-P, Callis PR, & Barkley MD (2006) Dependence of Tryptophan Emission Wavelength on Conformation in Cyclic Hexapeptides. The Journal of Physical Chemistry B 110:7009-7016.

90. Gallagher T, Alexander P, Bryan P, & Gilliland GL (1994) Two Crystal

G and Comparison with NMR. Biochemistry 33:4721-4729.

91. Ababou A & Bombarda E (2001) On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins. Protein Science 10:2102-2113.

92. Toptygin D, Gronenborn AM, & Brand L (2006) Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan. The Journal of Physical Chemistry B 110:26292-26302.

93. Calhoun DB, Vanderkooi JM, Holtom GR, & Englander SW (1986) Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure. Proteins: Structure, Function, and Bioinformatics 1:109-115.

94. Sheinerman FB & Brooks Iii CL (1998) Calculations on folding of segment B1 of streptococcal protein G. Journal of molecular biology 278:439-456.

95. Robson B & Pain RH (1971) Analysis of the code relating sequence to conformat ion in proteins: Possible implications for the mechanism o f formation of helical regions. Journal of molecular biology 58:237-257.

96. Dill KA (1985) Theory for the folding and stability of globular proteins.

Biochemistry 24:1501-1509.

97. Gutin AM, Abkevich VI, & Shakhnovich EI (1995) Is Burst Hydrophobic Collapse Necessary for Protein Folding? Biochemistry 34:3066-3076.

98. Dolgikh DA, Gilmanshin RI, Brazhnikov EV, Bychkova VE, Semisotnov GV, Venyaminov SY, & Ptitsyn OB (1981) α-lactalbumin: compact state with fluctuating tertiary structure? FEBS Letters 136:311-315.

99. Ohgushi M & Wada A (1983) Molten-globule state: a compact form of globular proteins with mobile side-chains. FEBS Letters 164:21-24.

100. Ptitsyn OB (1995) Molten Globule and Protein Folding. Advances in protein chemistry, eds C.B. Anfinsen FMRJTE & David SE (Academic Press), Vol Volume 47, pp 83-229.

101. Kuwajima K (1989) The molten globule state as a clue for understanding the folding and cooperativity of globular protein structure. Proteins: Structure, Function, and Bioinformatics 6:87-103.

附錄