螢光強度監控雷射捕陷控制溶菌酶的結晶化

Abstract

我們利用雷射捕陷的方式進而控制溶菌酶的結晶化，藉由監控隨著時間變化的螢光強度。我們運用近紅外光連續波當作雷射捕陷的光源，此光源聚焦於溶菌酶的緩衝溶液中，且此溶液中含有些許的螢光染料標定在溶菌酶上，在高濃度區域中含有溶菌酶團簇，此團簇遠大於聚焦點，可由周圍的雷射焦點的螢光強度增加得知。我們選擇從焦點離10 μm 水平面當作觀測點，此點螢光強度在起始溶液假設為1.0 倍，並觀測隨著時間變化的螢光強度而變化。在雷射照射期間中，不論照射時間多長，沒有發現雷射誘發任何現象，然而關掉雷射後，卻呈現出兩種現象分別是結晶化和液液相分離。這些現象主要依靠達到螢光強度所致，雷射誘發的現象能夠由螢光強度改變而控制。當螢光強度達到1.6倍時，立即停止雷射捕陷，觀察此現象均呈現一致性溶菌酶的結晶化。有趣的是，大部分的結晶密集出現在焦點周圍幾微米地方，由此可推測被誘發溶菌酶結晶是因為雷射捕陷而形成微米大小的高濃度區域所導致。這些現象也需要依賴雷射照射時間與極性甚至當達到一樣的螢光強度。另一個實例中，當螢光強度1.4倍時，雷射照射時會出現兩種情況，分別是沒有任何現象和結晶出現的機率。有趣的是，利用線性偏振的雷射照射時，在長時間照射優先導致結晶化，而右旋圓偏振卻是短時間照射優先導致結晶化。從結果上來說，我們考慮到觀察這些現象取決於不單單在於區域中的叢集濃度以及團簇間連結和方向，而取決於雷射平均照射時間和極性。因此，雷射捕陷的方式足以形成高濃度的溶菌酶叢集區域和能強烈限制叢集的聯結和方向，在焦點1 μm的位置。當停止雷射照射時，雷射所造成限制已消逝，使得在區域中的叢集能重新排列，並觸發結晶成核與液液相分離的現象。我們相信利用雷射捕陷的方式進行結晶化能夠應用各種蛋白質中，且在這次研究中觀察獨特的雷射誘發現象，同時也給予新的視野觀去了解和闡述他們的成核現象的機制。

We present laser trapping-controlled crystallization of hen egg white lysozyme (HEWL) by monitoring temporal change in fluorescence intensity. By applying the laser trapping of a focused near-infrared continuous-wave laser beam into HEWL buffer solution with a small amount of fluorescent dye-labelled HEWL, a highly-concentrated domain consisting of HEWL liquid-like clusters much larger than the laser focus is formed, according to which the fluorescence intensity around the laser focus is gradually increased. We select a point of 10 μm away horizontally from the laser focus as a monitoring point, where the fluorescence intensity of the initial solution is set to be 1.0, and monitor the temporal change in the fluorescence intensity. During the laser irradiation, no laser trapping-induced phenomenon is found even by long-time irradiation, while two kinds of phenomena of its crystallization and liquid-liquid phase separation are realized only after stopping the laser trapping. These phenomena strongly depend on the fluorescence intensity achieved by laser trapping, and trapping-induced phenomena can be controlled by monitoring the fluorescence intensity change. In the case that the laser trapping is stopped when the fluorescence intensity reaches 1.6, HEWL crystallization is consistently observed. Interestingly, the point of crystal generation is densely distributed just in a few millimeters around the focal spot, which supports that HEWL crystal is induced through the formation of a millimeter-sized highly concentrated domain by laser trapping. These phenomena also depend on laser irradiation time and polarization even when the achieved fluorescence intensity is same. In the case that the achieved fluorescence intensity is 1.4, laser irradiation causes nothing and crystallization with a certain probability. Interestingly, linearly-polarized laser irradiation preferentially leads to crystallization by long-term irradiation, while circularly-polarized laser irradiation does by short-term irradiation. Based on this result, we consider that the observed phenomenon is determined not simply by cluster concentration in the domain but also their association and orientation depending on laser average irradiation time and polarization. Thus, laser trapping can form a large highly concentrated domain of HEWL clusters and can strongly restrict the association and orientation of the clusters at the laser focus of about 1 μm. When the restriction is released by stopping laser trapping, the clusters in the domain can align suitable for triggering crystal nucleation and liquid-liquid phase separation. We believe that this crystallization method utilizing laser trapping can be applied to various kinds of proteins and that the unique laser trapping-induced phenomena observed in this work give a new sight to understand and elucidate their nucleation mechanism.