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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.


The signal-to-background was calculated as the fluorescence intensity in the presence of labeled Flu-KKK divided by the fluorescence intensity of buffer.
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Figure 2: The signal-to-background was calculated as the fluorescence intensity in the presence of labeled Flu-KKK divided by the fluorescence intensity of buffer.

Mentions: The optimal trace concentration was determined for each fluorescent-peptide. Each peptide was titrated and the fluorescence intensity measured. The ratio of fluorescence intensity to background was plotted versus tracer concentration. For optimal sensitivity, the fluorescently labeled peptide tracer concentration was set to produce a total fluorescence signal-to-background ratio greater than 50. For example, for Flu-ALLQ this required a concentration of 50 nM (data not shown) and 10 nM for Flu-KKK (Fig. 2).


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

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

The signal-to-background was calculated as the fluorescence intensity in the presence of labeled Flu-KKK divided by the fluorescence intensity of buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The signal-to-background was calculated as the fluorescence intensity in the presence of labeled Flu-KKK divided by the fluorescence intensity of buffer.
Mentions: The optimal trace concentration was determined for each fluorescent-peptide. Each peptide was titrated and the fluorescence intensity measured. The ratio of fluorescence intensity to background was plotted versus tracer concentration. For optimal sensitivity, the fluorescently labeled peptide tracer concentration was set to produce a total fluorescence signal-to-background ratio greater than 50. For example, for Flu-ALLQ this required a concentration of 50 nM (data not shown) and 10 nM for Flu-KKK (Fig. 2).

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.