A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein–DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

[1]  G. Bloom,et al.  Three‐color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells , 2015, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[2]  Andong Xia,et al.  Characterization of photoinduced isomerization and intersystem crossing of the cyanine dye Cy3. , 2007, The journal of physical chemistry. A.

[3]  Sabine Müller,et al.  Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity. , 2011, Journal of the American Chemical Society.

[4]  K. Schulten,et al.  Dynamic profiling of double-stranded RNA binding proteins , 2015, Nucleic acids research.

[5]  Sua Myong,et al.  Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions. , 2014, Chemical Society reviews.

[6]  Shimon Weiss,et al.  Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers , 2016, Scientific Reports.

[7]  A. van der Vaart,et al.  Photophysical and dynamical properties of doubly linked Cy3-DNA constructs. , 2014, The journal of physical chemistry. B.

[8]  B. Poolman,et al.  Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ , 2014, Nature Structural &Molecular Biology.

[9]  Shimon Weiss,et al.  Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. , 2006, The journal of physical chemistry. B.

[10]  M. F. White,et al.  Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach , 2015, Nucleic acids research.

[11]  Christoph Bräuchle,et al.  Pulsed interleaved excitation. , 2005, Biophysical journal.

[12]  A. van der Vaart,et al.  Cy3-DNA stacking interactions strongly depend on the identity of the terminal basepair. , 2011, Biophysical journal.

[13]  A Volkmer,et al.  Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. , 2001, Journal of biotechnology.

[14]  G. Cosa,et al.  Binding kinetics and affinities of heterodimeric versus homodimeric HIV-1 reverse transcriptase on DNA-DNA substrates at the single-molecule level. , 2013, The journal of physical chemistry. B.

[15]  Hajin Kim,et al.  Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity , 2011, Proceedings of the National Academy of Sciences.

[16]  CoSMoS Unravels Mysteries of Transcription Initiation , 2012, Cell.

[17]  L. Stryer,et al.  Energy transfer: a spectroscopic ruler. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Huber,et al.  Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence , 2014, Nucleic acids research.

[19]  Timothy D Craggs,et al.  Alternating-laser excitation: single-molecule FRET and beyond. , 2014, Chemical Society reviews.

[20]  C. Richardson,et al.  Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin , 2010, Proceedings of the National Academy of Sciences.

[21]  Stephen Mann,et al.  Quantitative sensing of microviscosity in protocells and amyloid materials using fluorescence lifetime imaging of molecular rotors , 2014, Photonics West - Biomedical Optics.

[22]  Volodymyr Kudryavtsev,et al.  Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements. , 2012, Chemphyschem : a European journal of chemical physics and physical chemistry.

[23]  Gerard Roelfes,et al.  Mechanism of intramolecular photostabilization in self-healing cyanine fluorophores. , 2013, Chemphyschem : a European journal of chemical physics and physical chemistry.

[24]  I. Z. Steinberg,et al.  Intramolecular dynamics of chain molecules monitored by fluctuations in efficiency of excitation energy transfer. A theoretical study. , 1984, Biophysical journal.

[25]  M. López-Quintela,et al.  Steric hindrance of diffusion controlled reactions , 1984 .

[26]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[27]  M. Sanborn,et al.  Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA. , 2007, The journal of physical chemistry. B.

[28]  S. Hamdan,et al.  Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA , 2010, Proceedings of the National Academy of Sciences.

[29]  P. V. von Hippel,et al.  Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments , 2014, Nucleic acids research.

[30]  Taekjip Ha,et al.  Cytosolic Viral Sensor RIG-I Is a 5'-Triphosphate–Dependent Translocase on Double-Stranded RNA , 2009, Science.

[31]  D. Rueda,et al.  Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I , 2012, Nucleic acids research.

[32]  Horst Wallrabe,et al.  Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation , 2012, Other Conferences.

[33]  Sanghwa Lee,et al.  Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time , 2010, PloS one.

[34]  J M Beechem,et al.  Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. , 1989, Biophysical journal.

[35]  Shimon Weiss,et al.  Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays. , 2012, Clinical chemistry.

[36]  Timothy D. Craggs,et al.  Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates , 2013, Nucleic acids research.

[37]  J. Lakowicz,et al.  Single-molecule and FRET fluorescence correlation spectroscopy analyses of phage DNA packaging: colocalization of packaged phage T4 DNA ends within the capsid. , 2010, Journal of molecular biology.

[38]  Nam Ki Lee,et al.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. , 2005, Biophysical journal.

[39]  D. Millar,et al.  Conformational distributions of a four-way DNA junction revealed by time-resolved fluorescence resonance energy transfer. , 1993, Biochemistry.

[40]  K. Y. Han,et al.  Effect of single-base mutation on activity and folding of 10-23 deoxyribozyme studied by three-color single-molecule ALEX FRET. , 2012, The journal of physical chemistry. B.

[41]  Nam Ki Lee,et al.  Alternating-laser excitation of single molecules. , 2005, Accounts of chemical research.

[42]  Brian A. Maxwell,et al.  Single-molecule Investigation of Substrate Binding Kinetics and Protein Conformational Dynamics of a B-family Replicative DNA Polymerase* , 2013, The Journal of Biological Chemistry.

[43]  T. Knowles,et al.  Molecular Rotors Provide Insights into Microscopic Structural Changes During Protein Aggregation. , 2015, The journal of physical chemistry. B.

[44]  J. Loparo,et al.  A general approach to visualize protein binding and DNA conformation without protein labelling , 2016, Nature Communications.

[45]  Boriana Marintcheva,et al.  A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Nam Ki Lee,et al.  Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. , 2007, Biophysical journal.

[47]  Monika A. Ciuba,et al.  Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3. , 2015, The journal of physical chemistry letters.

[48]  W. Nau,et al.  Diffusion-enhanced Förster resonance energy transfer and the effects of external quenchers and the donor quantum yield. , 2013, The journal of physical chemistry. B.

[49]  H. Viadiu,et al.  The role of metals in catalysis by the restriction endonuclease Bam HI , 1998, Nature Structural Biology.

[50]  Nam Ki Lee,et al.  Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules , 2004, Proceedings of the National Academy of Sciences of the United States of America.