Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.

Huff, Sheela; Matsuka, Yuri; McGavin, Martin J.; Ingham, Kenneth C.

doi:10.1016/s0021-9258(17)40717-4

Cited by 51 publications

(30 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 45 kDa fibronectin fragment has been shown to induce MMP-3 and 13 synthesis, via an IL-1-independent mechanism, and aggrecanase-mediated aggrecan degradation 58 . Three fibronectin fragments were used in the present study including the 29 kDa heparin binding fragment 59 (type I repeating units), the 45 kDa gelatin binding fragment 60 (consisting of type I and type II repeating units), and the 70 kDa heparin and gelatin binding fragment 61 . All three fragments, in a similar fashion to IL-1b induced TLR2 upregulation although the 45 kDa fragment appeared to have the greatest effect.…”

Section: Discussionmentioning

confidence: 99%

Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes

Tsai

Lee

et al. 2005

Osteoarthritis and Cartilage

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 99%

Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes

Tsai

Lee

et al. 2005

Osteoarthritis and Cartilage

View full text Add to dashboard Cite

show abstract

“…where the subscripts V and H refer to the orientation (vertical or horizontal) of the polarizers for the intensity measurements, with the first subscript indicating the position of the excitation polarizer and the second for the emission polarizer. The G factor is defined as the following equation ( 2): The resulting concentration-dependent change in anisotropy was fitted to the following equation (3): 14…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescence anisotropy has been an effective tool for molecular interaction studies. 6,[14][15][16] This method provides an easy and reliable way for studying binding between a labelled aptamer and a specific target and for quantitative analysis of the target protein. 6 As is shown in Fig.…”

Section: Real-time Detection Of Ang Using Fluorescence Anisotropymentioning

confidence: 99%

Aptamer-based analysis of angiogenin by fluorescence anisotropy

Wang

Tan

et al. 2007

Analyst

View full text Add to dashboard Cite

Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.

show abstract

“…Fig. 3 shows the competitive displacement data of 5 independent duplicate experiments for (sulfo)peptides R2A-D fitted to the equation of Huff et al 19 ; the K d values determined are listed in Table 1. The data show, as expected, that sulfation of tyrosine residues in peptides derived from the N-terminus of CCR2 enhances binding to MCP-1(P8A).…”

Section: Fluorescence Anisotropy Assays For Binding Of Proteins To Fl...mentioning

confidence: 99%

“…All competitive binding assays were performed in 50 mM MOPS, pH 7.0 with final sample volumes of 200 μL in each well. Duplicate assays were performed five times independently and the average data fitted by non-linear regression analysis using GraphPad Prism v.6.0 software to the equation for a 1 : 1 competitive displacement curve described by Huff et al 19 in which: the independent variable is the concentration of non-fluorescent (sulfo)peptide; the dependent variable is the observed anisotropy signal; fixed input parameters are the total concentrations of FL-R2D and MCP-1(P8A), the final anisotropy signal, which is the anisotropy of free FL-R2D, and the K d for 1 : 1 binding between FL-R2D and MCP-1(P8A), determined using the direct binding assay; and fitted parameters are the initial anisotropy signal and the K d for 1 : 1 binding of the non-fluorescent (sulfo)peptide to MCP-1(P8A).…”

Section: Fluorescence Anisotropy Binding Assaysmentioning

confidence: 99%

Phosphate modulates receptor sulfotyrosine recognition by the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2)

et al. 2015

View full text Add to dashboard Cite

Tyrosine sulfation is a widespread post-translational modification that mediates the interactions of secreted and membrane-associated proteins in such varied biological processes as peptide hormone action, adhesion, blood coagulation, complement activation and regulation of leukocyte trafficking. Due to the heterogeneous nature of tyrosine sulfation, detailed biochemical and biophysical studies of tyrosine sulfation rely on homogenous, synthetic sulfopeptides. Here we describe the synthesis of a fluorescent sulfopeptide (FL-R2D) derived from the chemokine receptor CCR2 and the application of FL-R2D in direct and competitive fluorescence anisotropy assays that enable the efficient measurement of binding affinities between sulfopeptides and their binding proteins. Using these assays, we have found that the binding of the chemokine monocyte chemoattractant protein-1 (MCP-1) to sulfated peptides derived from the chemokine receptor CCR2 is highly dependent on the assay buffer. In particular, phosphate buffer at close to physiological concentrations competes with the receptor sulfopeptide by binding to the sulfopeptide binding pocket on the chemokine surface. Thus, physiological phosphate may modulate the receptor binding selectivity of chemokines.

show abstract

Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.

Cited by 51 publications

References 34 publications

Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes

Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes

Aptamer-based analysis of angiogenin by fluorescence anisotropy

Phosphate modulates receptor sulfotyrosine recognition by the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2)

Contact Info

Product

Resources

About