Genetically encoded sensors of protein hydrodynamics and molecular proximity

Proc Natl Acad Sci U S A. 2015 May 19;112(20):E2569-74. doi: 10.1073/pnas.1424021112. Epub 2015 Apr 30.

Abstract

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

Keywords: FLIM; FRET; fluorescence anisotropy; fluorescent protein; protein sensor.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • DNA Primers / genetics
  • Escherichia coli
  • Fluorescence Polarization / methods*
  • Fluorescence Resonance Energy Transfer
  • Genetic Engineering / methods
  • Hydrodynamics
  • Image Processing, Computer-Assisted
  • Luminescent Proteins* / genetics
  • Luminescent Proteins* / metabolism
  • Microscopy, Confocal
  • Molecular Probes* / metabolism
  • Multiprotein Complexes* / genetics
  • Multiprotein Complexes* / metabolism
  • Plasmids / genetics
  • Proteins / analysis*
  • Thrombin / analysis
  • cdc42 GTP-Binding Protein / metabolism

Substances

  • Bacterial Proteins
  • DNA Primers
  • Luminescent Proteins
  • Molecular Probes
  • Multiprotein Complexes
  • Proteins
  • lumazine protein, Photobacterium
  • yellow fluorescent protein, Bacteria
  • Thrombin
  • cdc42 GTP-Binding Protein