Aim 1 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 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 specifically 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 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 HOLO state to the APO state. Without tmFRET-derived distance constraints, the simulations did not find the APO conformation of MBP. Simulations that contained the tmFRET-derived distances, however, 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.
Aim 2 Membrane proteins in cells exist in a complex molecular environment. For example, many membrane proteins assemble as complexes. Furthermore, dozens of binding partners may transiently interact with membrane proteins to modulate their behavior. Finally, the architecture of a protein is influenced by post-translational modifications and the native membrane environment. Understanding these complex structural parameters is necessary for understanding the function and regulation of membrane proteins. We are using FRET to map the structures of membrane proteins within native biological membranes. These studies will help us understand how these proteins are structured, how their complexes assemble, and how the structure of these complexes is regulated within living cells. In this aim, we have been observing the structure and conformational dynamics of the plasma membrane t-SNARE syntaxin. This membrane protein is a core component of the protein machinery responsible for fusing synaptic vesicles with the plasma membrane. Syntaxin has been proposed to adopt a closed inactive conformation and an open active conformation. To understand these transitions, we imaged the structure of syntaxin 1A in living cell membranes with fluorescence resonance energy transfer (FRET). Specifically, FRET between fluorescently-tagged syntaxin 1A and the membrane-resident FRET acceptor dipicrylamine (DPA) was used to map the relative distances between domains in syntaxin 1A and the plane of the plasma membrane. Our results map the architecture of syntaxin in both of these states relative to the membrane and have opened the door to determining the structures and structural transitions of membrane proteins in a complex cellular environment with FRET.

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National Heart, Lung, and Blood Institute
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Yu, Xiaozhen; Strub, Marie-Paule; Barnard, Travis J et al. (2014) An engineered palette of metal ion quenchable fluorescent proteins. PLoS One 9:e95808
Yu, Xiaozhen; Wu, Xiongwu; Bermejo, Guillermo A et al. (2013) Accurate high-throughput structure mapping and prediction with transition metal ion FRET. Structure 21:9-19
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