臺灣博碩士論文加值系統

利用傳統停止─流動裝置來觀測蛋白質摺疊動力學，一直受制於其過長的混合截止時間，並且變性劑的使用經常是不可避免的。事實上，變性劑對摺疊過程的影響一直未被討論。若變性劑解離及蛋白質摺疊是兩個速率相似的過程，則我們是無法分別這兩者的。此篇論文中，我們利用一種光不穩定化合物來偵測胜肽於奈秒至毫秒內的摺疊過程，此光不穩定化合物是一個有機的保護基或稱之為“籠”化合物。用於胜肽側鏈保護策略的稱為双甲氧─α─羥─α─苯基苯乙酮(Dimethoxy-benzoin: DMB)，而用來環化蛋白質N端到胜肽側鏈的稱為溴乙醯基─羧基甲氧─α─羥─α─苯基苯乙酮(bromoacetyl-carboxymethoxybenzoin: BrAc-CMB)，我們所用的胜肽都是由固相胜肽合成法所製造的。
我們試用不同條件與試劑合成DMB及Fmoc-Asp(DMB)-OH，並由紫外線光譜研究它們的光解特性。由圓二色光譜儀和二維核磁共振光譜儀所測得的資料，我們可以得知側鏈有保護基的胜肽之結構和其野生型之結構差異並不大，所以由DMB所保護的側鏈位置必須改變。環化蛋白質N端到胜肽側鏈策略有較大的潛能使蛋白質變性，我們合成了兩條環化的胜肽，並加以純化分離與鑑定。

The traditional stopped-flow methods for following the kinetic time course of protein folding reactions are limited by the dead-time of mixing. Moreover high concentrations of a denaturant are usually added to unfold proteins at the outset. In fact, how the denaturant affect folding pathways has been ignored. We cannot distinguish between the dissociation rate of denaturant and protein refolding rate. Using the photolabile linkers, a general method to monitor protein folding process shorter than 1 microsecond has been developed. The method is based on the photolysis of small organic protecting groups, or “cage” compounds. The compounds used in the studies are 3’,5’-dimethoxybenzoin (DMB) and bromoacetyl-carboxymethoxybenzoin (BrAc-CMB). These cage compounds are incorporated in to a three stranded β-sheet for the “sidechain” cage strategy and the N-terminal sequence of ubiquitin for “head-to-sidechain cyclization” strategy by Fmoc solid-phase peptide synthesis.
DMB and Fmoc-Asp(DMB)-OH were synthesized by different reactions and conditions. The properties of Fmoc-Asp(DMB)-OH and the “sidechain” caged peptide were further studied by UV spectroscopy. The “sidechain” caged peptide has no clear structural difference from its wildtype according to the CD spectroscopy and 2D-NMR data. So, the caged position had to be changed. The “cyclization” strategy has more potential to unfold the peptide. Two cyclized peptides were synthesized and purified.

Abbreviations i
Abstract iv
中文摘要 v
Chapter 1 Introduction 1
1. Protein folding problem 1
1.1 Significance of protein folding 1
1.2 Folding models 2
1.3 Kinetic studies of protein folding 6
1.3.1 Previous methodologies to study protein folding 6
(a) Stopped-flow, continuous-flow and quenched-flow 6
(b) Pressure jump 7
(c) NMR relaxation 8
1.4 Burst-phase in folding kinetics 8
1.4.1 Definition 8
1.4.2 Significance of early folding events 11
1.4.3 Methodologies for studying the early stage of protein folding 13
(a) Laser flash photolysis 14
(b) CO flash photolysis 14
(c) Photoreduction of cytochrome c heme (rapid electron transfer) 14
(d) Temperature-jump 15
(e) Ultrasonic absorption 16
1.4.4 Elementary steps in protein folding 17
1.5 Problems of using denaturant in folding studies 18
1.6 Our proposed new triggering method 20
1.7 Peptide models 25
1.8 Photoacoustic calorimetry 27
1.9 Aim of the project 29
Chapter 2 Materials and Methods 31
2.1 Materials 31
2.1.1 Water 31
2.1.2 Chemicals 31
2.1.3 Chromatography column, membranes and filters 34
2.1.4 Peptide automated synthesizer 35
2.1.5 Centrifugation 35
2.1.6 Peptide purification by HPLC 35
2.1.7 Lyophilizer 35
2.1.8 Mass spectrometry 36
2.1.9 Nuclear magnetic resonance spectroscopy 36
2.1.10 Ultraviolet and visible spectroscopy 37
2.1.11 Circular dicroism spectroscopy 37
2.2 Methods 37
2.2.1 Synthesis of 3’,5’-dimethoxybenzoin (DMB) 37
2.2.2 Synthesis of Fmoc-Asp(DMB)-OH 38
(a) Esterification (DCC/DMAP) 38
(b) Esterification (EDC/DMAP) 39
(c) Mitsunobu reaction 40
(d) Deprotection of allyl group 40
2.2.3 Purification of BrAc-CMB-OH 41
2.2.4 Solid phase peptide synthesis 41
2.2.6 Final cleavage 44
2.2.5 Head-to-side chain cyclization of peptide with BrAc-CMB-OH 45
2.2.7 Peptide purification and identification 46
Chapter 3 Results and Discussion (I): Side chain cage strategy 47
3.1 Synthesis of Fmoc-Asp(DMB)-OH 47
3.2 Characterization of Fmoc-Asp(DMB)-OH 55
3.3 Synthesis of 20-mer and 20-mer T9D 57
3.4 CD characteristics of 20-mer and 20-merT9D 58
3.5 Synthesis of 20-merT9D(DMB) 59
3.6 Characterization of 20-merT9D(DMB) 64
Chapter 4 Results and Discussion (II): Cyclization strategy 66
4.1 Purification of BrAc-CMB-OH 66
4.2 Synthesis of c-CMB-U(1-17)T9DE16KV17C (cyclized from 1 to17) 66
4.3 Synthesis of c-CMB-U(1-21)T9DE16KE18C (cyclized from 1 to 18) 70
Chapter 5 Conclusions and Future plans 73
References 74
Appendix 84
謝 誌 94

Anfinsen, C. B. (1973) Principles that govern the folding of protein chains. Science, 18, 223-230.
Baldwin, J. E., McConnaughie, A. W., Moloney, M. G. Pratt, A. T., and Shim, S. B. (1990) New photolabile phosphate protecting groups. Tetrahedron, 45, 6879-6884.
Ballew, R. M., Sabelko, J., and Gruebele, M. (1996) Direct observation of fast protein folding: the initial collapse of apomyoglobin. Proc. Natl. Acad. Sci. USA, 93, 5759-5764.
Ballew, R. M., Sabelko, J., and Gruebele, M. (1996) Observation of distinct nanosecond and microsecond folding event. Nat. Struct. Biol. 3, 923-926.
Bieri, O. and Kiefhber, T. (1999) Elementary steps in protein folding. Biol. Chem., 380, 923-929.
Bieri, O., Wirz, J. Hellrung, B., Schutkowski, M., Drewello, M., and Kiefhaber, T. (1999) The speed limit for protein folding measured by triplet-triplet energy transfer. Proc. Natl. Acad. Sci. USA, 96, 9597-9601.
Braslavsky, S. E., and Heibel, G. E. (1992) Time-resolved photothermal and photoacoustics methods applied to photoinduced processes in solution. Chem. Rev. 92, 1381-1410.
Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature, 416, 507-511.
Callender, R. H., Dyer, R. B., Gilmanshin, R., and Woodruff, W. H. (1998) Fast events in protein folding: the time evolution of primary processes. Annu. Rev. Phys. Chem., 49, 173-202.
Chen, E., Wood, M. J., Fink, A. L., and Kliger, D. S. (1998) Time-resolved circular dicroism studies of protein folding intermediates of cytochrome c. Biochemistry, 37, 5589-5598.
Chen, P.-Y., Lin, C.-K, Lee, C.-T. Jan, H., and Chan S. I. (2001) Effects of turn residues in directing the formation of the β-sheet and in the stability of the β-sheet. Protein Sci. 10, 1794-1800.
Chen, P.-Y., Gopalacushina, B. G., Yang, C.-C., Chan, S. I. and Evans, P. A. (2001) The role of a β-bulge in the folding of the β-hairpin structure in ubiquitin. Protein Sci., 10, 2063-2074.
Colón, W., Elöve, G. A., Wakem, L. P., Sherman, F., and Roder, H. (1996) Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. Biochemistry, 35, 5538-5549.
Corrie, J. E. T., and Trenthan, D. R. (1992) Synthesis, mechanistic and photochemical studies of phosphate esters of substituted benzoins. J. Chem. Soc. Trans. 1, 2409-2417.
Desai, G., Panick, G., Zein, M., Winter, R., and Royer, C. A. (1999) Pressure-jump studies of the folding/unfolding of trp repressor. J. Mol. Biol., 288, 461-475.
Dill, K. A. (1990) Dominant forces in protein folding. Biochemistry, 29, 7133-7155.
Dill, K. A., Fiebig, K. M., and Chan, H. S. (1993) Cooperativity in protein-folding kinetics. Proc. Natl. Acad. Sci. USA, 90, 1942-1946.
Dill, K. A., Bromberg, S., Yue, K., Fiebig, K. M., Yee, D. P., Thomas, P. D., and Chan, H. S. (1995) Principles of protein folding-a perspective from simple exact models. Protein Sci., 4, 561-602.
Ellis R. J., and Pinheiro, J. T. (2002) Danger-misfolding proteins. Nature, 416, 483-484.
Gilmanshin, R., Williams, S., Callender, R. H., Woodruff, W. H. and Dyer R. B. (1997) Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc. Natl. Acad. Sci. USA, 94, 3709-3713.
Givens, R. S., Jung, A., Park, C.-H., Weber, J. and Bartlett, W. (1997) New photoactivated protecting group. J. Am. Chem. Soc., 119, 8369-8370.
Gruenewald, B., Nicola, C. U., Lustig, A., and Schwarz, G. (1979) Kinetics of the helix-coil transition of a polypeptide with non-ionic side chain groups, derived from ultrasonic relaxation measurements. Biophys. Chem. 9, 137-147.
Hagen, S. J., Hofrichter, J. Szabo, A. and Eaton, W. A. (1996) Diffusion-limited contact formation in unfolded cytochrome c: Estimating the maximum rate of protein folding. Proc. Natl. Acad. Sci. USA, 93, 11615-11617.
Hansen, K. C., Rock, R. S., Larsen, R. W., and Chan, S. I. (2000) A method for photoinitiating protein folding in a nondenaturing environment. J. Am. Chem. Soc., 122, 11567-11568.
Haque, T. S., and Gellman S. H. (1997) Insights on β-hairpin stability in aqueous solution from peptides with enforced type I’ and type II’ β-turn. J. Am. Chem. Soc., 119, 2303-2304.
Huang, G. S., and Oas, T. G. (1995) Submillisecond folding of monomeric λ repressor. Proc. Natl. Acad. Sci. USA, 92, 6878-6882.
Jacob M., Holtermann, G., Perl, D., Reinstein, J., Schindler, T., Geeves, M. A., and Schmid, F. X. (1999) Microsecond folding of the cold shock protein measured by a pressure-jump technique. Biochemistry, 38, 2882-2891.
Jaenicke, R. (1999) Stability and folding of domain proteins. Progr. Biophys. Mol. Biol. 71, 155-241.
Jennings, P. A., and Wright, P. E. (1993) Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science, 262, 892-896.
Jones, C. M., Henry, E.R., Hu. Y., Chan, C-K., Luck, S. D., Bhuyan, A., Roder, H., Hofrichter, .J. and Eaton, W. A. (1993) Fast events in protein folding initiated by nanosecond laser photolysis. Proc. Natl. Acad. Sci. USA, 90, 11860-11864.
Karplus, M., and Weaver D. L. (1994) Protein folding dynamics: the diffusion-collision model and experimental data. Protein Sci., 3, 650-668.
Kim, P. S., and Baldwin, R. L. (1990) Intermediates in the folding reactions of small proteins. Ann. Rev. Biochem., 59, 631-660.
Kurzer, F., and Douraghi-Zadeh K. (1967) Advances in the chemistry of carbodiimides. Chem. Rev., 67, 107-152.
Kuwajima, K., Yamaya, H., Miwa, S., Sugai, S., and Nagamura, T. (1987) Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dicroism. FEBS Lett. 221, 115-118.
Leopold, P. E., Montal, M., and Onuchic. J. N. (1992) Protein folding funnel: A kinetic approach to the sequence-structure relationship. Proc. Natl. Acad. Sci. USA, 89, 8721-8725.
Muñoz, V., Thompson, P. A., Hofrichter J. and Eaton W. A. (1997) Folding dynamics and mechanism of β-hairpin formation. Nature, 390, 196-199.
Neises, B., and Steglich, W. (1978) Simple method for the esterification of carboxylic acids. Angew. Chem. Int. Ed. Engl., 17, 522-524.
Nölting, B., Golbik, R., and Fersht A. R. (1995) Submillisecond events in protein folding. Proc. Natl. Acad. Sci. USA, 92, 10668-10672.
Pascher, T., Chesick, J. P., Winkler, J. R., and Gray, H. B. (1996) Protein folding triggered by electro transfer. Science, 271, 1558-1560.
Park, S.-H., Shartry, M. C. R., and Roder, H. (1999) Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing. Nat. Struct. Biol. 6, 943-947.
Ptitsyn, O. B. (1995) How the molten globule became. Trends in Biochem. Sci., 20, 376-379.
Rock, R. S. and Chan, S. I. (1996) Synthesis and photolysis properties of a photolabile linker based on 3-methoxybenzoin. J. Org. Chem., 61, 1526-1529.
Rock, R. S. (1999) Thesis of Ph.D, Caltech.
Roder, H., and Shastry, MC. R. (1999) Methods for exploring early events in protein folding. Curr. Opin. Struct. Biol., 7, 620-626.
Sabelko, J., Ervin, J., and Gruebele, M. (1998) Cold-denatured ensemble of apomyoglobin: Implications for the early steps of folding. J. Phys. Chem. B, 102, 1806-1819.
Sabelko, J., Ervin, J., and Gruebele, M. (1999) Observation of strange kinetics in protein folding. Proc. Natl. Acad. Sci. USA, 96, 6031-6036.
Sauder, J. M., and Rode, H. (1998) Amide protection in an early folding intermediate of cytochrome c. Fold. Des., 3, 293-301.
Schenck H. L., and Gellman S. H. (1998) Use of a designed triple-stranded antiparallel β-sheet to prove β-sheet cooperativity in aqueous solution. J. Am. Chem. Soc., 120, 4869-4870.
Shastry, M. C. R., and Roder, H. (1998) Evidence for barrier-limited folding kinetics on the microsecond time scale. Nat. Struct. Biol., 5, 385-392.
Sheehan, J. C., and Wilson, R. M. (1964) Photolysis of desyl compounds. A new photolytic cyclization. J. Am. Chem. Soc., 86, 5277-5281.
Sheehan, J. C., Wilson, R. M. and Oxford, A. W. (1971) The photolysis of methoxy-substituted benzoin esters. A photosensitive protecting group for carboxylic acids. J. Am. Chem. Soc., 93, 7222-7228.
Shi, Y., Corrie, J. E. T., and Wan, P. (1997) Mechanism of 3’,5’-dimethoxybenzoin ester photochemistry: heterolytic cleavage intramolecularly assisted by the dimethoxybenzene ring is the primary photochemical step. J. Org. Chem., 62, 8278-8279.
Sosnick, T. R., Shtilerman, M. D., Mayne, L., and Englander, S. W. (1997) Ultrafast signals in protein folding and the polypeptide contracted state. Proc. Natl. Acad. Sci. USA, 94, 8545-8550.
Stowell, M. H. B., Rock, R. S., Rees, D. C., and Chan, S. I. (1996) Efficient synthesis of photolabile alkoxy benzoin protecting groups. Tet. Lett., 37, 307-310.
Szilágyi, L. (1995) Chemical shifts in proteins come of age. Progress in nuclear magnetic resonance spectroscopy, 27, 325-443.
Thompson, P., Eaton, W., and Hofrichter, J. (1997) Laser temperature jump study of the helix-coil kinetics of an alanine peptide interpreted with a “kinetic zipper” model. Biochemistry, 36, 9200-9210.
Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987) Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol., 194, 531-544.
Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term potentiation in vivo. Nature, 416, 535-539.
Wetlaufer, D. B. (1990) Nucleation in protein folding confusion of structure and process. Trends in Biochem. Sci., 15, 414-415.
Williams, S., Causgrove, P., Gilmanshin, R., Fang, K. S., Callender, R. H., Woodruff, W. H., and Dyer, R. B. (1996) Fast events in protein folding: helix melting and formation in a small peptide. Biochemistry, 35, 691-697.
Wittung-Stafshede, P., Malmström, B. G., Sanders, D., Fee, J. A., Winkler, J. R., and Gray, H. B. (1998) Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: The CuA domain of cytochrome oxidase from Thermus thermophilus. Biochemistry, 37, 3172-3177.
Wolynes, P. G., Onuchic, J. N., and Thirumalai, D. (1995) Navigating the folding routes. Science, 267, 1619-1620.
Wüthrich, K. (1986) NMR of proteins and nucleic acids, (John Wiley & Sons, New York).
Zerella, R., Evans, P. A., Ionides, J. M. C., Williams, D. H., Trotter, B. W., and Mackay, J. P. (1999) Autonomous folding of a peptide corresponding to the N-terminal β-hairpin from ubiquitin. Protein Sci., 8, 1320-1331.
Zerella, R., Chen, P. Y., Evans, P. A., Raine, A., and Williams, D. H. (2000) Structural characterization of mutant peptides derived from ubiquitin and implications for protein folding. Protein Sci., 9, 2142-2150.
Zhang, J., Peng, X., Jonas, A., and Jonas, J. (1995) NMR study of cold, heat, and pressure unfolding of ribonuclease A. Biochemistry, 34, 8631-8641.
Novobiochem 1999 catalog& peptide synthesis handbook.
PS3 automated solid phase peptide synthesizer guide book, Rainin instrument CO. Inc.