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Fluorescence strategies for high-throughput quantification of protein interactions.

Hieb AR, D'Arcy S, Kramer MA, White AE, Luger K - Nucleic Acids Res. (2011)

Bottom Line: Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function.We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies.In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT
Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A-H2B heterodimer.

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FRET analysis reveals salt-dependent binding of PARP1 to DNA. (A) A cartoon representation showing FRET as a result of protein–DNA interaction. (B) Representative microplate images showing the binding of donor-labeled nPARP1Donor to DNAAcceptor. Top three images are raw data showing acceptor, donor and FRET channels for acceptor only samples (A) and samples containing a donor–acceptor pair (DA). Fcorr is the FRET image which has been mathematically corrected for spectral overlap using ImageJ software; a high contrast image of Fcorr exemplifies the lack of signal in the DNAAcceptor only titration and that signal arises only from nPARP1Donor being present. (C) Representative binding curves showing the interaction between nPARPDonor to DNAAcceptor using FRET. Curves show binding reactions performed in 200 (red filled circles with solid lines) and 250 mM (black filled squares with solid lines) NaCl, respectively. Data were fit to a single binding curve (Equation 3). R2 values for shown curves meet or exceed 0.97. (D) A log–log plot of salt concentration versus binding affinity reveals a linear dependence on binding between nPARPDonor and DNAAcceptor, indicating an ionic dependence to binding. The data are fit to a line to extract the ionic dependence on binding. (E) Stoichiometric measurements of nPARPDonor to DNAAcceptor performed at elevated protein concentrations (200 nM) at 175 mM NaCl. Each of the three linear phases was fit to lines, with intersections indicating both 1:1 and 2:1 DNA:PARP stoichiometries. Points and error bars represent the average and range of two replicates.
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gkr1045-F3: FRET analysis reveals salt-dependent binding of PARP1 to DNA. (A) A cartoon representation showing FRET as a result of protein–DNA interaction. (B) Representative microplate images showing the binding of donor-labeled nPARP1Donor to DNAAcceptor. Top three images are raw data showing acceptor, donor and FRET channels for acceptor only samples (A) and samples containing a donor–acceptor pair (DA). Fcorr is the FRET image which has been mathematically corrected for spectral overlap using ImageJ software; a high contrast image of Fcorr exemplifies the lack of signal in the DNAAcceptor only titration and that signal arises only from nPARP1Donor being present. (C) Representative binding curves showing the interaction between nPARPDonor to DNAAcceptor using FRET. Curves show binding reactions performed in 200 (red filled circles with solid lines) and 250 mM (black filled squares with solid lines) NaCl, respectively. Data were fit to a single binding curve (Equation 3). R2 values for shown curves meet or exceed 0.97. (D) A log–log plot of salt concentration versus binding affinity reveals a linear dependence on binding between nPARPDonor and DNAAcceptor, indicating an ionic dependence to binding. The data are fit to a line to extract the ionic dependence on binding. (E) Stoichiometric measurements of nPARPDonor to DNAAcceptor performed at elevated protein concentrations (200 nM) at 175 mM NaCl. Each of the three linear phases was fit to lines, with intersections indicating both 1:1 and 2:1 DNA:PARP stoichiometries. Points and error bars represent the average and range of two replicates.

Mentions: Depending on the location of the fluorophore, not all interactions result in significant fluorescence (de)quenching. To overcome this, we have adapted the HI-FI assay for FRET. FRET is the distance-dependent non-radiative transfer of energy from an excited donor to an acceptor fluorophore, where the efficiency of transfer is strongly dependent on the distance between the two fluorophores (4). FRET is, therefore, observed only when an interaction occurs between distinct donor- and acceptor-labeled molecules (Figure 3A) (22). While the error of FRET experiments is usually small, they require fluorescent labeling of both binding partners.Figure 3.


Fluorescence strategies for high-throughput quantification of protein interactions.

Hieb AR, D'Arcy S, Kramer MA, White AE, Luger K - Nucleic Acids Res. (2011)

FRET analysis reveals salt-dependent binding of PARP1 to DNA. (A) A cartoon representation showing FRET as a result of protein–DNA interaction. (B) Representative microplate images showing the binding of donor-labeled nPARP1Donor to DNAAcceptor. Top three images are raw data showing acceptor, donor and FRET channels for acceptor only samples (A) and samples containing a donor–acceptor pair (DA). Fcorr is the FRET image which has been mathematically corrected for spectral overlap using ImageJ software; a high contrast image of Fcorr exemplifies the lack of signal in the DNAAcceptor only titration and that signal arises only from nPARP1Donor being present. (C) Representative binding curves showing the interaction between nPARPDonor to DNAAcceptor using FRET. Curves show binding reactions performed in 200 (red filled circles with solid lines) and 250 mM (black filled squares with solid lines) NaCl, respectively. Data were fit to a single binding curve (Equation 3). R2 values for shown curves meet or exceed 0.97. (D) A log–log plot of salt concentration versus binding affinity reveals a linear dependence on binding between nPARPDonor and DNAAcceptor, indicating an ionic dependence to binding. The data are fit to a line to extract the ionic dependence on binding. (E) Stoichiometric measurements of nPARPDonor to DNAAcceptor performed at elevated protein concentrations (200 nM) at 175 mM NaCl. Each of the three linear phases was fit to lines, with intersections indicating both 1:1 and 2:1 DNA:PARP stoichiometries. Points and error bars represent the average and range of two replicates.
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Related In: Results  -  Collection

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gkr1045-F3: FRET analysis reveals salt-dependent binding of PARP1 to DNA. (A) A cartoon representation showing FRET as a result of protein–DNA interaction. (B) Representative microplate images showing the binding of donor-labeled nPARP1Donor to DNAAcceptor. Top three images are raw data showing acceptor, donor and FRET channels for acceptor only samples (A) and samples containing a donor–acceptor pair (DA). Fcorr is the FRET image which has been mathematically corrected for spectral overlap using ImageJ software; a high contrast image of Fcorr exemplifies the lack of signal in the DNAAcceptor only titration and that signal arises only from nPARP1Donor being present. (C) Representative binding curves showing the interaction between nPARPDonor to DNAAcceptor using FRET. Curves show binding reactions performed in 200 (red filled circles with solid lines) and 250 mM (black filled squares with solid lines) NaCl, respectively. Data were fit to a single binding curve (Equation 3). R2 values for shown curves meet or exceed 0.97. (D) A log–log plot of salt concentration versus binding affinity reveals a linear dependence on binding between nPARPDonor and DNAAcceptor, indicating an ionic dependence to binding. The data are fit to a line to extract the ionic dependence on binding. (E) Stoichiometric measurements of nPARPDonor to DNAAcceptor performed at elevated protein concentrations (200 nM) at 175 mM NaCl. Each of the three linear phases was fit to lines, with intersections indicating both 1:1 and 2:1 DNA:PARP stoichiometries. Points and error bars represent the average and range of two replicates.
Mentions: Depending on the location of the fluorophore, not all interactions result in significant fluorescence (de)quenching. To overcome this, we have adapted the HI-FI assay for FRET. FRET is the distance-dependent non-radiative transfer of energy from an excited donor to an acceptor fluorophore, where the efficiency of transfer is strongly dependent on the distance between the two fluorophores (4). FRET is, therefore, observed only when an interaction occurs between distinct donor- and acceptor-labeled molecules (Figure 3A) (22). While the error of FRET experiments is usually small, they require fluorescent labeling of both binding partners.Figure 3.

Bottom Line: Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function.We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies.In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT
Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A-H2B heterodimer.

Show MeSH
Related in: MedlinePlus