Protein biosynthesis at the single-molecule level in live cells

该奖项由化学部门的生命过程化学颁发。来自威斯康星大学麦迪逊分校的Silvia Cavagnero和James Weisshaar将使用单分子荧光显微镜来测量一个核糖体生物合成一个蛋白质分子所需的时间。迄今为止，人们对活细胞中复杂的化学和生物物理事件（包括基因表达）的时间进程知之甚少，这主要是由于在散装溶液中进行的实验的解释所固有的困难。为了实现提案的目标，dr。Cavagnero和Weisshaar将利用含有荧光团的非天然氨基酸，并通过单个琥珀色密码子在很早的时候将其纳入感兴趣的特定基因序列中。这项研究将产生新的见解，以分布的翻译率感兴趣的基因在活的细菌细胞。在与新生蛋白结合后，荧光团标记的非天然氨基酸将非常缓慢地扩散，直到从核糖体中释放。在携带荧光标签的完全合成蛋白释放后，荧光团的扩散将会迅速得多。单个相机帧的定量分析将能够以大约一秒的精度确定新生蛋白质在核糖体上的停留时间。我们将首先研究单结构域蛋白HmpH和相关的三结构域蛋白Hmp的体内合成，以测试和优化新方法。其他多结构域蛋白的翻译速率也可以被研究。最终目标是开发一套新的生物学和光谱工具，以了解停顿位点如何影响活细胞中单个基因的翻译时间，并了解这些停顿如何分布在不同的翻译核糖体中。这种新方法有望为活细胞中许多其他多步骤生化事件的未来研究开辟机会，包括折叠、共/翻译后修饰和各种酶促反应。蛋白质，即活细胞的主要成分，逐步积累所需的时间对细胞的健康和健康功能至关重要。如果一种蛋白质合成太慢，在细胞死亡之前，它可能永远无法为细胞的活动做出贡献。相反，如果蛋白质合成太快，它可能永远无法达到正确的三维形状，因为快速合成没有足够的时间进行适当的三维折叠。因此，这种蛋白质可能聚集并不可逆转地损害细胞的功能。总之，很明显，细胞中蛋白质生物合成的时间必须恰到好处，即严格调节正确的细胞功能。这个项目的目标是测量活细菌细胞中蛋白质合成的速率，一次一个分子。考虑到体内合成过程的复杂性，单分子方法是测量合成时间的唯一方法。每个蛋白质都将直接在活细胞中通过荧光标签“点亮”。这种新颖的方法将为高分辨率探索细胞功能和功能失调的起源提供前所未有的机会。因此，这项研究将使博士。Cavagnero和Weisshaar研究了地球上生命最重要的过程之一——蛋白质生物合成的时间过程。该项目将为学生和博士后提供高度跨学科的机会，他们将在开展本研究的同时协同整合生物学，荧光和显微镜的概念。Drs。Cavagnero和Weisshaar将非常积极地鼓励女性、未被充分代表的少数民族和本科生参与这项研究，追求科学事业。他们还将共同开展一项新的毕业生招聘活动，名为“化学机会”（CHemistry OPportunitieS）。一些对化学感兴趣的大三学生和大四学生将在秋季的一个周末访问威斯康星大学麦迪逊分校。通过为参观者提供动手操作的单分子显微镜讲习班，研究人员将把这项研究与更广泛的目标联系起来，即在化学和生物学的界面上加强被忽视的少数民族对科学的参与。

In this award from the Chemistry of Life Processes in the Chemistry Division, Drs. Silvia Cavagnero and James Weisshaar, from the University of Wisconsin, Madison, will use single-molecule fluorescence microscopy to measure the time required for the biosynthesis of one single protein molecule by one ribosome. The timecourse of complex chemical and biophysical events in living cells, including gene expression, is poorly understood to date, largely due to the difficulties inherent in the interpretation of experiments performed in bulk solution. Towards the goal of the proposal, Drs. Cavagnero and Weisshaar will exploit unnatural amino acids bearing a fluorophore, and engineered to be incorporated very early in the sequence of specific genes of interest via single amber codons. This study will yield novel insights into the distribution of the translation rates of genes of interest within live bacterial cells. Upon incorporation into the nascent protein, the fluorophore-labeled unnatural amino acid will diffuse very slowly until release from the ribosome. After release of the fully synthesized protein carrying the fluorescent tag, diffusion of the fluorophore will be much more rapid. Quantitative analysis of individual camera frames will enable the determination of the residence time of the nascent protein on the ribosome with about one-second accuracy. The in vivo synthesis of the single-domain protein HmpH and the related three-domain protein Hmp will be studied first, to test and optimize the novel methodology. The translation rates of other multi-domain proteins may also be investigated. The ultimate goal is to develop a novel set of biological and spectroscopic tools to understand how pause sites affect the translation time of individual genes in live cells, and to learn how these pauses are distributed across different translating ribosomes. This novel approach is expected to open up opportunities for future studies of many other multi-step biochemical events in live cells, including folding, co-/post-translational modifications, and a wide variety of enzymatic reactions.The time required for the stepwise buildup of proteins, i.e., the main components of living cells, is essential for the cell's well being and healthy function. If a protein is made too slowly it may never be able to contribute to cell's activity before the cell dies. Conversely, if a protein is made too fast it may never be able to achieve its correct three-dimensional shape because the fast synthesis does not allow enough time for proper three-dimensional folding. The protein may thus aggregate and irreversibly compromise the cell's function. In summary, it is clear that the timing of protein biosynthesis in the cell has to be just right, i.e., tightly regulated for correct cell function. The goal of this project is to measure the rate of protein synthesis in living bacterial cells, one molecule at a time. The single-molecule approach is the only way to measure synthesis times, given the complexity of the process in vivo. Each protein will be 'lit up' via labeling with a fluorescent tag directly in the living cell. This novel approach will open up unprecedented opportunities to explore the origin of cell's function and misfunction at high resolution. This study will thus enable Drs. Cavagnero and Weisshaar to interrogate the timecourse of one of the most important processes for life on earth, protein biosynthesis. This project will offer highly interdisciplinary opportunities to students and postdocs, who will synergistically integrate concepts from biology, fluorescence and microscopy, while carrying out this research. Drs. Cavagnero and Weisshaar will be very active in encouraging women, underrepresented minorities and undergraduate students to participate in this research and pursue careers in science. Together, they will also develop a new graduate recruitment event called CHOPS, standing for CHemistry OPportunitieS. A few college juniors and seniors interested in chemistry will come to visit the University of Wisconsin-Madison for a weekend in the fall. By providing hands-on single-molecule microscopy workshop for the visitors, the investigators will tie this research to the broader goal of enhancing underrepresented minority participation in science at the interface of chemistry and biology.