Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed biochemical activities connected by rapidly diffusing second messengers, metabolic intermediates, ions or other proteins. It will probably remain within the realm of genetics to identify the ensemble of proteins that constitute these functional units and to establish the first-order connectivity. The dynamics of interactions within these protein machines can be assessed in living cells by the application of fluorescence spectroscopy on a microscopic level, using fluorescent proteins that are introduced within these functional units. Fluorescence is sensitive, specific and non-invasive, and the spectroscopic properties of a fluorescent probe can be analysed to obtain information on its molecular environment. The development and use of sensors based on the genetically encoded variants of green-fluorescent proteins has facilitated the observation of 'live' biochemistry on a microscopic level, with the advantage of preserving the cellular context of biochemical connectivity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing correlation with phenomena such as the cell cycle, migration and morphogenesis.
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