Genetically encoded sensors of protein hydrodynamics and molecular proximity

• DOI: 10.1073/pnas.1424021112
• Publication Date: 2015-05-01T01:22:32Z
• Authors: Alexander C. Hoepker; Ariel Wang; Alix Le Marois; Klaus Suhling; Yuling Yan; Gerard Marriott
• Keywords: 570; FLIM; Image Processing; Fluorescence Polarization; fluorescence anisotropy; 03 medical and health sciences; Computer-Assisted; Bacterial Proteins; Protein sensor; Escherichia coli; Fluorescence Resonance Energy Transfer; Image Processing, Computer-Assisted; fluorescent protein; cdc42 GTP-Binding Protein; DNA Primers; Microscopy; 0303 health sciences; Microscopy, Confocal; Thrombin; Proteins; Fluorescent protein; protein sensor; Luminescent Proteins; Confocal; Molecular Probes; Multiprotein Complexes; FRET; Hydrodynamics; Genetic Engineering; Fluorescence anisotropy; Plasmids

Significance The lumazine binding protein (LUMP) emits a cyan-colored fluorescence and has the longest average fluorescence lifetime of any genetically encoded fluorescent protein complex. Coupled with a small mass of 20 kDa, LUMP and its fusion with capture sequences are exploited as unique sensors of protein hydrodynamics and are shown to enable quantitative fluorescence anisotropy imaging of specific target proteins in vitro and in vivo. Moreover, the surface location of the lumazine probe is shown to improve the efficiency of Förster resonance energy transfer (FRET) with the Venus acceptor protein compared with CFP, which is used in the development of a new class of FRET-based sensor.