Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

Chen, Yan; Müller, Joachim D.

doi:10.1073/pnas.0606557104

“…1C). This result agrees with an earlier study where it was shown that the NID fragment of steroid receptor coactivator-1, which contains the three NR boxes, is able to bind three RXRLBD (9). Whereas the earlier study was limited to a coactivator fragment, the present data show the same surprising result for the full-length coactivator.…”

Section: Resultssupporting

confidence: 93%

“…Our experiments identify a nontrivial 3∶1 stoichiometry between RXRLBD and TIF-2 in the presence of agonist (Fig. 1C), which agrees with a previous study limited to the NID fragment of a related coactivator (9). If both proteins are full-length, a change in the stoichiometry occurs with two RXR bound to the coactivator TIF-2 (Fig.…”

Section: Discussionsupporting

confidence: 91%

“…A single-color brightness technique for determining the stoichiometry of heteroprotein complexes under stoichiometric binding conditions has been described (9). The protein expression ratio that leads to the highest single-color brightness identifies the stoichiometry of the heteroprotein complex.…”

Section: Discussionmentioning

confidence: 99%

“…We previously demonstrated that all three NR boxes of the NID fragment of the coactivator can be occupied by RXRLBD (Fig. 1C) (9). Because the binding of three NRs to a coactivator is unexpected, it raises the question whether this result is a consequence of using protein fragments.…”

mentioning

confidence: 97%

“…Single-color brightness analysis utilizes this effect to characterize the binding curve and the stoichiometry of protein homocomplexes directly from cellular data (5). An extension of single-color brightness analysis for the identification of protein heterocomplexes has been described (8,9), but the applicability of the technique is very limited.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

Wu

¹

,

Chen

²

,

Müller

³

2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions, but lacks suitable methods for quantifying protein heterointeractions in the cell. We address this challenge by introducing heterospecies partition (HSP) analysis for protein heterointeractions of the type D þ nA ↔ DA n . HSP directly identifies the heterointeracting species from the sample mixture and determines the binding curve and stoichiometry of the protein complex. The HSP method is applied to provide the first direct characterization of the ligand-dependent binding of the retinoic X receptor to the coactivator transcription intermediate factor 2. A previous study based on protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein heterointeractions by dual-color fluorescence fluctuation spectroscopy in living cells. Brightness characterizes the average fluorescence intensity of a single particle and encodes the stoichiometry of a protein complex (5-7). If two fluorescently labeled monomers form a homodimer, the fluorescence intensity of the particle and therefore its brightness doubles. Single-color brightness analysis utilizes this effect to characterize the binding curve and the stoichiometry of protein homocomplexes directly from cellular data (5). An extension of single-color brightness analysis for the identification of protein heterocomplexes has been described (8, 9), but the applicability of the technique is very limited.The desire to study heterointeractions motivated the introduction of dual-color FFS (10, 11). Unlike homointeractions, heterointeractions between two proteins D and A require differently colored labels to distinguish the proteins. Each of the two detection channels primarily receives the signal from one of the two colored labels. Dual-color FFS retains color-information of a species by measuring its brightness in each of the two detection channels. Thus, dual-color brightness analysis could in principle reveal the binding curve and stoichiometry of heteroprotein interactions, because each protein species (such as D, A, DA, DA 2 ) has its own distinct dual-color brightness. Unfortunately, a general analysis method for extracting this information from dual-color brightness data is not available. Because of this limitation, dual-color brightness analysis is currently restricted to the detection of protein heterointeractions without the ability to quantify the reaction.We overcome this shortcoming of the dual-color brightness technique by introducing heterospecies partition (HSP) analysis. HSP isolates the h...

show abstract

A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures

Duckworth

¹

,

Chen

²

,

Wollack

³

et al. 2007

Self Cite

View full text Add to dashboard Cite

DNA has numerous attractive features as a scaffold for nanostructure assembly. Its rigidity, predictable structure, and assembly through complementary hybridization allow DNA to form nanoscale architectures such as cubes, [1] tetrahedra, [2] octahedra, [3,4] and 2D arrays. [5][6][7][8] By introducing proteins into DNA nanostructures, the recognition elements and functionalities that are inherent in proteins can be organized into nanostructured motifs. DNA-scaffolded protein assemblies have been used in immuno-PCR detection methods (PCR = polymerase chain reaction) [9][10][11] to arrange biocatalysts in a series for sequential reactions [12,13] and to organize other nanomaterials. [14] There are currently several methodologies used to link proteins to DNA. Proteins have been assembled onto DNA scaffolds through intervening adapter molecules such as streptavidin [12,[15][16][17][18][19][20] or aptamers. [21,22] Alternately, direct covalent conjugation can be achieved by modification of cysteine or lysine residues [23][24][25][26] or intein modification. [11,27,28] Niemyer and co-workers have employed these protein-DNA conjugates to form fluorescence resonant energy transfer (FRET) systems for use in nanobiotechnology. [29,30] Herein, we demonstrate a fusion-based strategy to regioselectively and covalently label proteins at the C terminus with single-stranded DNA. These protein-oligonucleotide chimeras were then spontaneously assembled into nanoarchitectures by complementary hybridization of the DNA. The covalent attachment strategy described herein yields a short and compact linkage between the protein and DNA molecule that allows for precise control over protein spacing and orientation in the final nanostructure.To achieve selective protein labeling, we use the enzyme protein farnesyltransferase (PFTase) to label a substrate protein containing a C-terminal tetrapeptide tag with an azide-modified isoprenoid diphosphate (1, Scheme 1).

show abstract

A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures

Duckworth

¹

,

Chen

²

,

Wollack

³

et al. 2007

Self Cite

View full text Add to dashboard Cite

DNA has numerous attractive features as a scaffold for nanostructure assembly. Its rigidity, predictable structure, and assembly through complementary hybridization allow DNA to form nanoscale architectures such as cubes, [1] tetrahedra, [2] octahedra, [3,4] and 2D arrays. [5][6][7][8] By introducing proteins into DNA nanostructures, the recognition elements and functionalities that are inherent in proteins can be organized into nanostructured motifs. DNA-scaffolded protein assemblies have been used in immuno-PCR detection methods (PCR = polymerase chain reaction) [9][10][11] to arrange biocatalysts in a series for sequential reactions [12,13] and to organize other nanomaterials. [14] There are currently several methodologies used to link proteins to DNA. Proteins have been assembled onto DNA scaffolds through intervening adapter molecules such as streptavidin [12,[15][16][17][18][19][20] or aptamers. [21,22] Alternately, direct covalent conjugation can be achieved by modification of cysteine or lysine residues [23][24][25][26] or intein modification. [11,27,28] Niemyer and co-workers have employed these protein-DNA conjugates to form fluorescence resonant energy transfer (FRET) systems for use in nanobiotechnology. [29,30] Herein, we demonstrate a fusion-based strategy to regioselectively and covalently label proteins at the C terminus with single-stranded DNA. These protein-oligonucleotide chimeras were then spontaneously assembled into nanoarchitectures by complementary hybridization of the DNA. The covalent attachment strategy described herein yields a short and compact linkage between the protein and DNA molecule that allows for precise control over protein spacing and orientation in the final nanostructure.To achieve selective protein labeling, we use the enzyme protein farnesyltransferase (PFTase) to label a substrate protein containing a C-terminal tetrapeptide tag with an azide-modified isoprenoid diphosphate (1, Scheme 1).

show abstract

Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

Cited by 59 publications

References 19 publications

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures

A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures

Contact Info

Product

Resources

About