High-throughput screen for Escherichia coli heat shock protein 70 (Hsp70/DnaK): ATPase assay in low volume by exploiting energy transfer.
J Biomol Screen
; 15(10): 1211-9, 2010 Dec.
Article
en En
| MEDLINE
| ID: mdl-20926844
ABSTRACT
Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70's weak enzymatic activity have combined to create significant challenges in high-throughput screening. To overcome these difficulties, the authors have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal Biochem 2005;342254-259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, the authors tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z' ~0.6, coefficient of variation ~8%), and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70 family members.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas HSP70 de Choque Térmico
/
Proteínas de Escherichia coli
/
Ensayos Analíticos de Alto Rendimiento
Idioma:
En
Revista:
J Biomol Screen
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos