Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Abstract

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports shortrange distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.fluorescence ͉ peptides ͉ FRET F luorescence resonance energy transfer (FRET) occurs when excitation energy is transferred from a donor fluorophore to an acceptor dye through weak dipole-dipole resonance interactions (1, 2). FRET is extremely sensitive to the distance between the two dyes, with the transfer rate inversely proportional to the sixth power of the distance between them. This steep distance dependence has suggested that FRET could be used as a spectroscopic ruler on the molecular scale (3, 4). In addition to distance, five other factors influence the rate of energy transfer: (i) the overlap of the donor's and acceptor's excitation and absorbance spectra, respectively; (ii) the refractive index of the solvent; (iii) the relative orientation of the dyes; (iv) the quantum yield of the donor; and (v) the extinction coefficient of the acceptor.Although FRET is sensitive to the distance between probes, several factors have prevented the widespread use of FRET to easily map backbone distances within proteins. First, most commonly used FRET pairs have R 0 values (distance at which FRET efficiency is 50%) between Ϸ30 and 60 Å and are only able to measure distances between Ϸ20 and 80 Å. Thus, to accurately measure distances, the probes must be separated by large distances relative to the size of many proteins. Also, the large size of many dyes and their long attachment linkers position fluorophores far from the backbone of the protein. These flexible linkers also increase the conformational space sampled by the dye (4, 5). Becau...

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