偵測快速摺疊動力學：藉由對光不穩定的包覆策略和雷射光解裝置來研究抗凍蛋白RD1的折疊動力學

Probing the Fast Folding Kinetics:Folding Studies of an Antifreeze Protein RD1 by Using Photolabile Caging Strategy and Laser Flash Photolysis

許淳傑(Chun-Chieh Hsu)

指導教授 ： 陳佩燁

國立臺灣大學/生命科學院/生化科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00257

摘要

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。

關鍵字

摺疊 ； RD1 ； 包覆策略 ； 聲波熱卡度計 ； 動力學

並列摘要

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).

並列關鍵字

folding ； RD1 ； cage ； photolabile ； photoacoustic calorimetry ； kinetics

參考文獻

Eaton, W.A., Muñz, V., Thompson, P.A., Chan, C.-K. and Hofrichter, J. (1997) Submillisecond kinetics of protein folding. Current Opinion in Structural Biology, 7, 10-14.

Anfinsen, C.B. and Haber, E. (1961) Studies on the reduction and re-formation of protein disulfide bonds. Journal of Biological Chemistry, 236, 1361-1363.

Arabaci, G., Guo, X.-C., Beebe, K.D., Coggeshall, K.M. and Pei, D. (1999) α-Haloacetophenone derivatives as photoreversible covalent inhibitors of protein tyrosine phosphatases. Journal of the American Chemical Society, 121, 5085-5086.

Avery, O.T., MacLeod, C.M. and McCarty, M. (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Inductions of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. The Journal of Experimental Medicine, 79, 137-158.

Baneyx, F. and Mujacic, M. (2004) Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology, 22, 1399-1408.