陳佩燁臺灣大學:生化科學研究所許淳傑Hsu, Chun-ChiehChun-ChiehHsu2010-05-042018-07-062010-05-042018-07-062009U0001-0408200912301400http://ntur.lib.ntu.edu.tw//handle/246246/178863為了進一步了解蛋白質摺疊，所以我們必須要對於摺疊的過程進行系統性的研究。我們利用蛋白質包覆策略（caging-strategy），並結合時間解析的聲波熱卡度計（PAC，photoacoustic calorimetry）研究RD1蛋白質（分子量7 kDa）的摺疊過程──包含摺疊的動力學、焓的變化及體積的變化。在蛋白質包覆策略策略中，為了在RD1序列中創造適合結合的取代基，我們利用定點突變技術將第七號胺基酸（丙氨酸；Ala，alanine）突變為半胱氨酸（Cys，cysteine）（突變後蛋白質縮寫：RD1-A7C），並且利用易受光分解的分子（4-(bromomethyl）-6,7-imethoxycoumarin，BrDMC）與半胱氨酸殘基結合（RD1-A7C-DMC），而破壞蛋白質的摺疊。此外，蛋白質的結構則利用核磁共振（NMR，Nuclear Magnetic Resonance）及圓二色光譜（CD，circular dichroism）技術分析。聲波熱卡度計以一道紫外光雷射（約10-9秒）打斷RD1-A7C-DMC中蛋白質與DMC之間的鍵結，同時引發蛋白質摺疊。由聲波熱卡度計的實驗結果發現，RD1-A7C的摺疊具有兩個過程：第一步為快速的體積收縮（volume contraction），摺疊的時間為20奈秒，並伴隨著-9.7 mL / mol的體積變化；接著為結構上的重新排列（comformational rearrangement），摺疊的時間約為470奈秒，同時伴隨著-1.4 mL / mol的體積變化。In order to understand the intrinsic principle of protein folding, events of the fold-ing process have to be systematically explored. In this work, the folding information of a small protein (RD1, about 7 kDa) including kinetic, enthalpy and volume change were reported by combining the photo-triggered caging-strategy and time-resolved photo-acoustic calorimetry. This strategy required the mutation with Ala-7 to Cys (designated RD1-A7C) that was introduced to incorporate a photolabile cage group, 4-(bromomethyl)-6,7-dimethoxycoumarin, to unfold the protein. The structural proper-ties of the caged protein were analyzed by nuclear magnetic resonance spectroscopy (NMR) and circular dichroism spectroscopy (CD). A pulse UV laser (~10-9 s) was used to break the photolabile cage and two events were observed in the refolding of RD1-A7C toward its native state by using photoacoustic calorimetry (PAC). The fast event, which has a folding time of 20 ns and a volume change of -9.7 mL/mol, was ex-plained as the result of rapid volume contraction. This event was followed by a con-formational rearrangement, which has a folding time ~ 470 ns and small volume change (-1.4 mL/mol).Contentsbbreviations ibstract iv文摘要 vhapter 1 Introduction 1.1 Introduction 1.2 Protein Folding 2.2.1 Driving force of protein folding 3.2.2 Protein folding models 4.3 Significance in protein folding 7.3.1 The role of protein folding 9.3.2 The diseases related to protein misfolding 10.4 The study of early events in protein folding 11.5 Ideal target proteins 13.5.1 RD1 15.6 Denaturation and initiating folding process 17.6.1 Photolabile cage strategy 18.6.2 2-Nitrobenzyl(NB) caged compounds 22.6.3 p-Hydroxyphenacyl (pHP) caged compounds 23.6.4 Coumarin-4-ylmethyl caged compounds 24.7 Observation of protein folding 27.7.1 Laser photoacoustic spectroscopy 28hapter 2 Materials and Methods 32.1 Materials 32.1.1 Water 32.1.2 Chemicals 32.1.3 Centrifuge 35.1.4 Membrane, filters 35.1.5 Circular dichroism spectroscopy (CD) 35.1.6 Electrophoresis 36.1.7 Gel filtration chromatography 36.1.8 High performance liquid chromatography (HPLC) 36.1.9 Ion exchange chromatography 36.1.10 Lyophilizer 37.1.11 Mass spectroscopy 37.1.12 Photochemical reactor 37.1.13 Ultraviolet spectroscopy 38.1.14 photoacoustic calorimetry (PAC) 38.1.15 Nuclear magnetic resonance (NMR) 38.1.16 Peptide synthesizer 39.2 Methods 40.2.1 Large scale over-expression and purification of RD1-A7C in the soluble form 40.2.2 Synthesis of peptide 42.2.3 Synthesis of caged protein 43.2.5 The proteins and the peptides identification 43.2.5 Photolysis of caged-protein and caged-peptide 44.2.6 Probing the proteins conformational change 44.2.7 PAC signals of caged-peptide and caged-protein 46hapter 3 Results (Ⅰ) 48.1 Large scale over-expression, purification and identification of RD1-A7C 48.2 Synthesis, purification and identification of RD1-A7C-DMC 53.3 Synthesis, purification and identification of caged-peptide 56hapter 4 Results ( Ⅱ ) 59.1 Examining the conformational change within secondary structure of protein after cage-addition by CD 59.2 Examining the conformational change within tertiary structure of protein after cage-addition by NMR spectroscopy 61.3 Photolysis of caged-protein 63.4 Examining the conformational change within secondary structure of protein after photolysis by CD 68.5 Refolding kinetics of the peptide and protein by photoacoustic calorimetry 70hapter 5 Discussion 75.1 RD1-A7C is a compact protein with a cavity, but without characteristic secondary structures 75.2 RD1-A7C-DMC is unfolded, but spontaneously refolds by photolysis 76.3 Two distinct events within caged protein photolysis 77.4 Enthalpy change in the refolding of RD1-A7C 80.5 The first step: fast volume contraction 82.6 The second step: structural rearrangement 83.7 Two-step folding model 85.8 BrDMC is an ideal cage group for caging-strategy 86hapter 6 Conclusions 87eferences 88application/pdf3595282 bytesapplication/pdfen-US摺疊RD1包覆策略聲波熱卡度計動力學foldingcagephotolabilephotoacoustic calorimetrykineticshttp://ntur.lib.ntu.edu.tw/bitstream/246246/178863/1/ntu-98-R96b46022-1.pdf