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

Abstract

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).