Improved Photostable FRET-Competent Biarsenical-Tetracysteine Probes

CARLA C. SPAGNUOLO, RUDOLF J. VERMEIJ, AND ELIZABETH A. JARES-ERIJMAN

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

Año: 2006 vol. 128 p. 12040 - 12041

0002-7863

Biarsenical probes are membrane-permeable fluorogenic dyes, which form highly stable complexes with tetracysteine motifs engineered into a target protein of interest. The probes and targets are small compared to visible fluorescent proteins, thereby reducing potential stereochemical interference while providing (i) controllable times of delivery in pulse chase experiments, and (ii) special reactivities enabling chromophore-assisted light inactivation experiments (CALI) and nonfluorescent readouts. Furthermore, the combination of biarsenical dyes with visible fluorescence proteins (VFPs) as Fo¨rster resonance energy transfer3 donor-acceptor (DA) pairs constitutes a very attractive technology for the assessment of conformational changes and molecular interactions in living cells. Due to the inverse 6th power distance dependence of FRET, the range of separations that can be determined with confidence about the Fo¨rster critical distance for 50% FRET efficiency (Ro) is very narrow. The generation of DA pairs with large Ro is essential for extending the range over which FRET is operative. Although optimization of the biarsenical binding motif has led to significant improvements in affinity and signal levels, the limited photostability and pH sensitivity of fluorescein derivatives in the physiological range constitute inherent limitations that still preclude their widespread application. Here we introduce fluoro-substituted versions, F2FlAsH and F4FlAsH, exhibiting significant improvements in important properties over the original fluorescein derivative FlAsH. Compared to FlAsH, F2FlAsH has higher absorbance, larger Stokes shift, higher quantum yield, higher photostability, and reduced pH dependence. The emission of F4FlAsH lies in a region intermediate to that of FlAsH and ReAsH (a resorufin biarsenical), providing a new color and excellent luminosity. In addition, the two new probes form a new FRET pair with a substantially larger Ro value than any obtained with these dyes.