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Development of fluorescence polarization assays for the molecular chaperone Hsp70 family members: Hsp72 and DnaK.

Ricci L, Williams KP - Curr Chem Genomics (2008)

Bottom Line: The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases.Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects.Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases. We were interested in developing a generally applicable assay format for the Hsp70 family and have developed fluorescence polarization based assays for both the mammalian Hsp72 and its bacterial counterpart, DnaK. These assays are comparable in assay set-up, incubation conditions and buffer components. Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects. Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72. We would predict that these assays will be suitable for identifying both selective chemical probes of Hsp70 family members and "pan" Hsp70 inhibitors.

No MeSH data available.


Various concentrations of Hsp72 protein were incubated with the Flu-HLA peptide (20 nM) and FP values measured.
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Figure 7: Various concentrations of Hsp72 protein were incubated with the Flu-HLA peptide (20 nM) and FP values measured.

Mentions: Another set of peptides have been identified as Hsp70 binders. Peptides derived from the MHC class I antigens have been shown to bind to Hsc70 and also have immunosuppressive properties [25,26]. We tested one of these peptides from HLA-B2702 (residues 75-84; RENLRIALRY; Table 1) in an FP assay. This 10-mer peptide was labeled on the N-terminus with fluorescein (termed Flu-HLA) and used as a tracer to measure Hsp72 binding (Fig. 7).


Development of fluorescence polarization assays for the molecular chaperone Hsp70 family members: Hsp72 and DnaK.

Ricci L, Williams KP - Curr Chem Genomics (2008)

Various concentrations of Hsp72 protein were incubated with the Flu-HLA peptide (20 nM) and FP values measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803438&req=5

Figure 7: Various concentrations of Hsp72 protein were incubated with the Flu-HLA peptide (20 nM) and FP values measured.
Mentions: Another set of peptides have been identified as Hsp70 binders. Peptides derived from the MHC class I antigens have been shown to bind to Hsc70 and also have immunosuppressive properties [25,26]. We tested one of these peptides from HLA-B2702 (residues 75-84; RENLRIALRY; Table 1) in an FP assay. This 10-mer peptide was labeled on the N-terminus with fluorescein (termed Flu-HLA) and used as a tracer to measure Hsp72 binding (Fig. 7).

Bottom Line: The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases.Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects.Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases. We were interested in developing a generally applicable assay format for the Hsp70 family and have developed fluorescence polarization based assays for both the mammalian Hsp72 and its bacterial counterpart, DnaK. These assays are comparable in assay set-up, incubation conditions and buffer components. Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects. Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72. We would predict that these assays will be suitable for identifying both selective chemical probes of Hsp70 family members and "pan" Hsp70 inhibitors.

No MeSH data available.