Aim Fluorescence resonance energy transfer (FRET), in which light energy absorbed by a donor is transferred to a nearby acceptor, is a powerful tool for measuring changes in molecular distances. The efficiency of FRET falls off with the sixth power of the distance between the two molecules, making FRET very sensitive to changes in distance. However, FRET can measure distances effectively only in a narrow range of distances that are not always well suited to study intra-molecular movements in proteins. We are developing rapid high throughput methods that use transition metal ions (nickel and copper) as energy acceptors for small fluorescent donor dyes (bimane) to map the conformational rearrangements of engineered proteins. These transition metal ion FRET (tmFRET) fluorescent methods work over shorter distances than classical FRET, use smaller dyes with shorter linkers, and are not as sensitive to the orientation problems usually associated with other methods. In this work, we have used tmFRET to map 10 unique distances in the model protein Maltose Binding Protein (MBP) in both the ligand-bound (HOLO) and ligand-free (APO) state. We have mapped distances between two donor dyes (monobromo-bimane and fluorescein-5-maleimide) and the two acceptor metals (nickel and copper). This has given us a total of 40 independent distance measurements in MBP. When these distances were compared to the x-ray crystal structure of MBP our tmFRET distances match the x-ray crystal structure to within a few angstroms. Furthermore, tmFRET was able to accurately detect structural changes in the protein during ligand binding. With the above experimental data, we next tested if distances derived with tmFRET could be used to guide molecular dynamics simulations. In these tmFRET-constrained simulations, MBP was allowed to move from the ligand-bound state to the APO state. Without tmFRET-derived distance constraints, the simulations did find the APO conformation of MBP. Simulations that contained the tmFRET-derived distances rapidly adopted the APO state. We conclude that tmFRET can be used to drive the conformational folding of proteins structures to an accuracy of a few angstroms. We have continued to develop this method as applied to other proteins. We have explored new metals including zinc and cobalt for binding and fluorescence effects in these systems. We have also developed new structural models of the angstrom-scale architecture of these systems and how they can be used to map other proteins in vitro or in vivo. These systems have been tested in living cells. Thus, tmFRET can be applied to proteins in a living cell environment to map the conformational dynamics of proteins in vivo at the angstrom scale.
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