The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their wide-spread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches en-abling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take ad-vantage of new chemistries for site-specific protein labeling. A great deal has changed since it was first shown that fluorescent proteins could be injected into living cells and studied in their native environment (1,2). The technique ini-tially proved its great value in studies of cytoskeletal proteins, where the dynamics of large multimolecular assemblies was critical to understanding function. However, this approach was the purview of specialists who found it worthwhile to isolate, fluorescently label, and reinject proteins, then analyze their dynamics with expensive, and often self-constructed, microscope systems and software. Only some proteins could survive such treatment with biological activity intact, and the specialized cameras and computer equipment re-quired to take full advantage of the fluorescent protein analogs were expensive and far less capable than todays tools. A veritable revolution was ushered in by the discovery of the green fluorescent protein (GFP) from the jellyfish Aequoria victoria (3–6). GFP not only opened the door to fluorescent fusion proteins that could be generated through relatively accessible, reproducible cloning and transfection procedures, but spurred the development of more affordable cameras, software, and complete 'turnkey' imaging systems to take advantage of the exploding interest in fluorescent analog cytochemistry. This in turn led to tremendous improvements in the capabilities of the equipment, bringing the technique very much into the mainstream, where it has become an important means to study the mechanisms of many funda-mental cellular processes. The original fluorescent analogs were purposefully designed with fluorophores that would not respond to their environ-ment, enabling precise quantitation of subcellular concentra-tions. The field is now reaching beyond such tagging to analogs whose fluorescence changes to report protein activ-ity. This was initially made possible through covalent labeling of proteins with dyes that shift their fluorescence spectra in response to changes in their protein environment (7,8), but has since been extended through the application of fluores-cence resonance energy transfer (FRET) using GFP or dyes (9 – 11). As new approaches to fluorescent reporter proteins were developed, biophysical and optical techniques evolved in parallel to derive more information from intracellular fluorescence, even for proteins tagged with fluorophores that do not respond to environmental changes. Better ways to label proteins site-specifically and introduce dye-labeled proteins into living cells are enabling us to better use the varied capabilities of dyes as environmental reporters. Here we will give a brief overview of these developments. Rather than focus on the rapidly expanding list of applications where tagged proteins have been used to follow protein localization, we will describe new fluorescent protein ap-proaches that reveal changing protein structure or ligand binding. The exciting new biophysical techniques that are partner to these new fluorescent protein analogs must also be set aside in this review. We hope this overview will provide a useful starting point for those wishing to harness these newly accessible techniques, or develop approaches of their own. Fluorescent Analog Cytochemistry-Tagging Proteins to Quantify Changing Localization In Vivo The great majority of recent fluorescent protein analogs have been made using GFP rather than covalent labeling with dye (Figure 1, Scheme 1). The ability to clone and transfect fluorescent proteins is not only more convenient, but pro-vides access to proteins that cannot be isolated and labeled or reintroduced into their native environment, including mem-brane-spanning proteins, or many targeted to cellular com-partments. The original wild-type GFP protein was greatly improved through alteration for mammalian codon usage and 755 Hahn and Chamberlain introduction of point mutations to enhance brightness and photostability (12–15). The tremendous range of GFP appli-cations, described in other reviews (16–20), attests to the value of the approach.
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