Visualization of Cellular Processes via Selective tagging of Proteins in vivo

Erwin Schrödinger Fellowship J 1993 Selective tagging of proteins in vivo Doris RIETHER 09.10.2000 As the complete map of the human genome has become available, medicinal science embarks on an entirely new era of complex challenges related to understanding the roles of proteins in the context of thousands of interrelated cellular components. In order to succeed, novel tools will be required to detect and visualize the key cellular components and their interactions in living cells with spatial and temporal resolution. The development of fluorescent sensors for any selected protein would have a tremendous impact in this regard. The visualization and tracking of the fates of individual proteins will then be possible, as will the mapping of entire signaling networks (simultaneous detection of a number of proteins). This project will focus on the visualization of cellular processes via selective tagging of proteins in vivo. We propose the construction of new probes enabling detection of proteins and their interactions in living cells. These fluorogenic probes are based on selective and cell environment-compatible reactions between the protein and the probe leading to selective protein tagging. Furthermore, designed probes, themselves non-fluorescent, become fluorescent when attached to the protein. Thus, we will test this concept on Human Carbonic Anhydrase 1, which will be selectively modified with an azide group (e.g. by selective alkylation). The fluorescent tag will then be introduced by means of the Staudinger reaction, which will first be tested in vitro Mowed by studies in vivo.