螢光蛋白雷射捕陷結晶化動力學：藉由非線性顯微成像研究光壓誘發相分離及晶核形成

Abstract

本計畫包含創新的光壓誘發蛋白質結晶生成法並且同時藉由多模態的影像/光譜 顯微術來研宄晶體生成時動力學的方法。

在第一年，我們會建構一個具有觀測多模態非線性影像能力的雷射捕陷(laser trapping)顯微系統:雙光子激發螢光影像(two-photon excitation fluorescence, TPEF)以及二 階非線性影像Second-Order Nonlinear Imaging, SONI)。我們選擇綠色螢光蛋白（green fluorescent protein , GFP)以及GFP的突變體作為一個模型系統，並且藉由TPEF/SONI來 觀察藉由光壓誘發結晶中液體-液體相分離（liquid-liquid phase separation, LLPS)的過 程。同時利用時間解析三維 TPEF(Time-resolved three-dimensional TPEF imaging)影像可 以觀測GFP在雷射捕陷光壓所造成的區域溶液濃度變化以及GFP分子在雷射捕陷焦點的 移動分布。關於LLPS/droplet的形成，結合寬域影像(wide field imaging)及3D TPEF影 像，我們可以得到在雷射捕陷結晶過程中所蘊含的動力學之重要資訊。

第二年，首先我們會專注於經由SONI觀測GFP在雷射捕陷結晶過程中所形成的 微小晶核。SONI提供了更高的靈敏度和更好的解析度以偵測大約100nm的晶核。同時， 我們將會偵測GFP結晶所產生的二倍頻訊號(second harmonic generation)做為晶核初 形成的指標。結合S0NI以及TPEF結果，我們可以深入的分析光壓誘發結晶過程：TPEF 偵測雷射捕陷誘發晶核的形成中濃度的變化；SONI則提供LLPS及近一步晶體成長之 資訊。我們將會討論雷射捕陷結晶成長的速率，所形成的結晶也會經由晶體分析來研宄 及比較相對於自然結晶相似與差異性。

第三年，我們預計檢驗各種其餘的蛋白質如鐵蛋白，耐熱行直接溶血素，肌紅蛋 白，細胞色素C、胰島素、清蛋白、肌動蛋白、原肌球蛋白、病毒外套膜蛋白和膜蛋白 等。對於蛋白質結晶研宄，蛋白質溶液中的液體-液體相分離是很重要的一項議題。而 這項研宄結果將能對於化學領域，結晶化學領域，生化領域等等造成相當大的衝撃。因 此，這些研宄結果對於雷射光鉗結晶中的雷射誘發結晶研宄是相當重要的。

Here applicant proposes a novel photon pressure-induced crystallization methodology for protein crystal generation through local liquid-liquid phase transition (LLPS) and direct investigation of LLPS enhanced nucleation and crystal growth dynamics with multimodal nonlinear imaging/spectroscopy.

First year, we will set up laser trapping microscope system that has multimodal nonlinear imaging ability. Established microscope enables simultaneous two-photon excitation fluorescence (TPEF) and Second-Order Nonlinear Imaging (SONI) measurement of protein crystallization process. We choose green fluorescent protein (GFP) and its mutants as a model protein for the observation of photon pressure induced crystallization through LLPS and dense liquid droplet formation by TPEF imaging and SONI. Time-resolved three-dimensional TPEF imaging of GFP under laser trapping will give an information of photon pressure induced local concentration change and fluctuation around the focus of the trapping laser. Results of wide field imaging of LLPS/droplet formation during laser trapping and 3D TPEF image will give important information to elucidate the dynamics of laser-induce local phase transition and following crystallization.

Second year, first we will focus to the observation of primary tiny crystal embryo generated during the laser trapping by SONI using GFP. SONI gives higher sensitivity and better resolution for the detection of primary crystalline assembly formation than conventional linear microscopic imaging methods. We can detect a formation of nucleus from it size of around 100 nm. We will investigate the laser trapping crystallization dynamics by detecting SHG generated from GFP to observe very primary crystal embryo formation. We will combine SONI and TPEF result to comprehensively understand the relation of photon pressure induced concentration change and fluctuation during nucleation.

We will also examine trapping laser induced enhancement of crystal growth. Anisotropic directional growth under trapping laser focusing near by the crystal shows the obvious enhancement of crystal growth rate. However rapidly grown part of the crystal is not necessary guaranteed to be the same crystalline structure as well as original part before enhancement. Thus we conduct a crystallographic study of the crystals grown under enhancement to determine the similarity/difference between original and grown part of the crystal.

Third year, we will examine the laser trapping crystallization of various other proteins such as ferritin, thermo-stable direct hemolysin, myogrobin, cytochrome C, insulin, albumin, action, toropomyosin, coat protein of the virus, and membrane protein, and so on. Primary nucleation and crystal growth process of all proteins will be examined TPEF/SONI multimodal imaging system to clarify the crystallization dynamics of these proteins under laser trapping. It will be a good demonstrate of capability of our laser trapping crystallization microscope system for the detection of crystal generation with high sensitivity and high spatial resolution. LLPS occurring in the protein solution is important issue for achieving in-situ crystallization of protein. The results should provide an impact on chemistry, crystal chemistry, bioscience, biochemistry, etc. Thus, all the results will be important for the development of the mechanism of the crystallization in the laser trapping crystallization.