雷射捕陷誘發高濃度叢集區域控制溶菌酶結晶化

Abstract

我們的主要研究目標是利用雷射捕陷誘發高濃度叢集區域控制溶菌酶結晶化和討論其結晶化之動力學和結晶機制。首先，將一連續波長之近紅外光雷射聚焦於溶菌酶溶液中並於聚焦點捕陷溶菌酶叢集。接著，雷射使焦點附近之叢集濃度上升後，藉由叢集之間的相互作用力，造成叢集區域增大並且向周圍生長，最終我們成功誘發具毫米尺寸的高濃度叢集區域。在此區域中，叢集的濃度、排列和剛性程度會受到照射雷射之能量、極性和時間所影響。我們利用其高濃度叢集區域給予溶菌酶新的成核環境。我們也成功證實透過調控雷射參數而控制溶菌酶晶體於此區域之形貌與結晶性。在原理上，我們的方法應該能應用於所有的蛋白質結晶化中，如相當難結晶化之膜蛋白。除此之外，我們也利用螢光標記之溶菌酶和螢光光譜去證實雷射捕陷生成高濃度之叢集區域誘發溶菌酶結晶化的動力學和機制。並藉由X 光分析得知雷射誘發溶菌酶之結晶品質優於或同等於自然生成之溶菌酶結晶。於我們我的研究結果和認知上，此研究於晶體學研究中是相當新穎且迷人的，同時，我們也透過雷射捕陷技術開啟了新的研究領域和新的方法控制蛋白質結晶化。

We present the first demonstration of laser trapping-induced nucleation of hen egg white lysozyme (HEWL). When a continuous-wave near-infrared laser beam is focused into HEWL buffer solution, a millimeter-sized highly concentrated domain of the liquid-like clusters is formed around the focus. The clusters in the formed domain are strongly controlled by laser trapping at the focus and exhibit high ordering and rigidity which inhibit crystal nucleation in the domain. Actually, the nucleation has been never induced during the laser irradiation. After turning off the trapping laser, the ordering and rigidity of the cluster are relaxed. As a result, the crystals are generated densely in a few millimeter area around the focus, which is consistent with the domain size. The morphology of HEWL crystals is also much different from that on spontaneous nucleation, and the crystal quality evaluated by single X-ray crystallography is as well or better. Since the mechanism involving such a domain formation should be general for proteins, this nucleation method using laser trapping can be in principle applied to all proteins. We believe that this novel method will open a new door for controlling protein nucleation precisely.