Current Chemical Genomics and Translational Medicine

2008, 2 : 90-95
Published online 2008 December 30. DOI: 10.2174/1875397300802010090
Publisher ID: CCGTM-2-90

RESEARCH ARTICLE
 Development of Fluorescence Polarization Assays for the Molecular Chaperone Hsp70 Family Members: Hsp72 and DnaK

Laura Ricci and Kevin P Williams, *
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA

* Address correspondence to this author at the Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA; Tel: 919-530-7726; Fax: 919-530-6600; E-mail: kpwilliams@nccu.edu

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.