Single molecule assay and in vitro expression of myosin-GFP fused protein

在单分子水平上观察分子的运动和功能使我们能够了解单个分子的动力学，而传统的方法给出了蛋白质分子所在系统的平均值。然而，用荧光染料化学标记蛋白质有可能破坏蛋白质的微观结构，从而改变蛋白质的功能。为了减少这个问题，我们考虑用生物荧光分子GFP（绿色荧光蛋白）标记蛋白质。(1)体外蛋白质合成系统的建立利用传统的基因表达系统制备具有生物活性的重组骨骼肌肌球蛋白亚片段1（S1）是不合适的。体外蛋白质合成法能够制备出具有生物学功能的S1与GFP融合蛋白。S1-GFP的个体ATP周转率，所有 关于我们 我们通过观察荧光ATP类似物Cy 3-ATP的缔合-（水解）-解离来测定蛋白质的功能。（2）通过GFP以外的荧光蛋白进行单分子鉴定使用其他荧光分子（绿色荧光蛋白突变体EYFP、海胆衍生的红色荧光蛋白DsRed），在低背景全内反射荧光显微镜下可以清楚地观察单个单分子。(3)弗雷廷测定分子间相互作用方法的建立为了验证荧光蛋白标记方法的有效性，对DNA促旋酶B二聚体之间的相互作用进行了评价。使用香豆素作为接头分子，形成一对GyrB-CFP和GyrB-YFP或一对GyrB-GFP和GyrB-DsRed。荧光共振能量转移（FRET）在溶液中的每一对中观察到，没有任何干扰，与常规方法标记的分子一样清晰。总之，用GFP或其突变体标记的蛋白质适于在单分子水平上观察。该方法有望在单分子水平上广泛应用于蛋白质进入细胞的动力学研究。少

The observation of the molecular motion and function at single molecule level allows us to know the dynamics of individual molecules, whereas the conventional methods give average values of the system where protein molecules are placed. However, chemically labeling proteins with fluorescent dyes has the potential to damage protein microstructure, which could in turn alter protein function. To minimize this problem, we have considered to label proteins with biofluorescent molecules, GFP (green fluorescent protein).The achievements of this study for these two years are listed below.(1) Development of in vitro protein synthesis system for preparing labeled proteinsThe gene expression system using conventional methods were found inadequate for the preparation of recombinant skeletal muscle myosin subfragment-1 (S1) in the biologically active form. In vitro protein synthesis was able to produce the fusion protein of S1 and GFP with biological functions. Individual ATP turnover by S1-GFP all … More ow us to assay the function of a protein by visualizing association-(hydrolysis)-dissociation of the fluorescent ATP analogue, Cy3-ATP.(2)Single-molecular identification by fluorescent proteins other than GFPUsing other fluorescent molecules (green fluorescent protein mutant EYFP, sea urchin derived red fluorescent protein DsRed), individual single molecules were clearly visualized under low background total internal reflection fluorescence microscopy.(3) Development of a method for measuring molecular interaction by FRETIn order to validate the method for fluorescent protein labeling, an interaction between DNA gyrase B dimer was assessed. Using coumermysin as a linker molecule, a pair of GyrB-CFP and GyrB-YFP or a pair of GyrB-GFP and GyrB-DsRed was formed. FRET was observed in each pair in solution without any interference as clear as do the molecules labeled by the conventional methods.In conclusion, proteins labeled with GFP or its mutants are appropriate for observation at single molecular level. This method is expected to widely apply to research the protein dynamics into the cell at single-molecular level. Less