Mechanisms of Stability of Robust Chaperones from Hyperthermophiles
Abstract
Recombinant hyperthermophile HSP60s were fully characterized in vitro. The recombinant Pfu HSP60 has optimal ATPase activity at 90C and pH of 7.0 and from 300 to 500 mM KCI Biacore (SPR) binding assays showed that the denatured target proteins could be loaded on to immobilized chaperones, and suggesting possible applications of affinity refolding of non-native proteins following recombinant expression. HSP60 is an effective ATP-dependent protein foldase even in 1-4 M guanidine chloride at 90C. Binding of denatured lysozyme conferred higher thcrmo-siahility on the chaperonin. and increased the optimal ATPase temperature from 90C to 100C. The HSP60 and sHSP were found to be extremely effective at disrupting stable cross-beta structures in prion amyloid proteins. Transient treatment with 2M-guanidine resulted in a modified HSP60 with 30% lower basal ATP hydrolysis but unaltered folding capacity. This suggests that recombinant HSP60, resembling the native HSP60. can be engineered by reassembly following exposure to hot guanidine.
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 03, 2009
- Accession Number
- ADA586573
Entities
People
- Frank T. Robb
Organizations
- University of Maryland, Baltimore