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

在单分子水平上对分子运动和功能的观察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，用荧光染料对蛋白质进行化学标记可能会破坏蛋白质的微观结构，进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。这两年的研究成果如下：(1)制备标记蛋白的体外蛋白质合成系统的建立传统方法的基因表达系统不足以制备具有生物活性的重组骨骼肌肌球蛋白亚段-1(S1)。体外蛋白质合成可产生具有生物学功能的S1和GFP融合蛋白。按S1-gfp计算的个人atp营业额均为…(2)荧光蛋白的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)，在低背景全反射荧光显微镜下可以清楚地显示单个单分子。(3)建立一种用Fretin测量分子相互作用的方法为了验证荧光蛋白标记方法的有效性，对DNA旋转酶B二聚体之间的相互作用进行了评估。以香豆素为连接物，形成一对GyrB-CFP和GyrB-YFP或一对GyrB-GFP和GyrB-DsRed。结果表明，GFP或其突变体标记的蛋白质适合于单分子水平的观察。该方法有望在单分子水平上广泛应用于细胞内蛋白质动力学的研究。较少

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