Protein biosynthesis at the single-molecule level in live cells

在化学部生命过程化学奖中，来自威斯康星州大学麦迪逊的Silvia Cavagnero和James Weisshaar博士将使用单分子荧光显微镜来测量一个核糖体合成一个蛋白质分子所需的时间。活细胞中复杂的化学和生物物理事件的时程，包括基因表达，迄今为止知之甚少，主要是由于在散装溶液中进行的实验的解释固有的困难。为了实现该提案的目标，Cavagnero博士和Weisshaar博士将利用带有荧光团的非天然氨基酸，并通过单个琥珀密码子将其很早就整合到感兴趣的特定基因序列中。这项研究将对活细菌细胞内感兴趣基因的翻译速率分布产生新的见解。当掺入新生蛋白质时，荧光团标记的非天然氨基酸将非常缓慢地扩散，直到从核糖体释放。在释放携带荧光标签的完全合成的蛋白质之后，荧光团的扩散将快得多。单个相机帧的定量分析将使得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。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，以测试和优化新的方法。也可以研究其他多结构域蛋白的翻译速率。最终目标是开发一套新的生物和光谱工具，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。这种新型方法预计将为未来研究活细胞中许多其他多步骤生化事件开辟机会，包括折叠、共翻译/翻译后修饰以及各种酶反应。蛋白质逐步积累所需的时间，即，作为活细胞的主要成分，它对细胞的健康和健康功能至关重要。如果一种蛋白质的合成太慢，它可能永远无法在细胞死亡之前为细胞的活性做出贡献。相反，如果蛋白质合成得太快，它可能永远无法获得正确的三维形状，因为快速合成没有足够的时间进行正确的三维折叠。因此，蛋白质可能聚集并不可逆地损害细胞的功能。总之，很明显，细胞中蛋白质生物合成的时机必须恰到好处，即，严格控制细胞功能。该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。每种蛋白质都将通过直接在活细胞中标记荧光标签来“点亮”。这种新方法将为高分辨率探索细胞功能和功能障碍的起源提供前所未有的机会。因此，这项研究将使Cavagnero和Weisshaar博士能够询问地球上生命最重要的过程之一--蛋白质生物合成的时间进程。该项目将为学生和博士后提供高度跨学科的机会，他们将在开展这项研究的同时协同整合生物学，荧光和显微镜的概念。Cavagnero博士和Weisshaar博士将积极鼓励妇女、代表性不足的少数民族和本科生参与这项研究，并从事科学事业。他们还将共同开发一个新的毕业生招聘活动，名为CHOPS，代表CHembersopportunitieS。一些对化学感兴趣的大学三年级和四年级学生将在秋天的一个周末来威斯康星大学麦迪逊分校参观。通过为参观者提供实践单分子显微镜研讨会，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代表性不足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.