Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate.
To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying beta-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of beta-glucosidase at 90 degrees C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90 degrees C with an overall DeltaG degrees of approximately 20 kcal mol(-1). The high temperatures needed to chemically denature P. furiosus beta-glucosidase and the large DeltaG degrees of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of beta-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.
Powers
Sara Lawrence
SL
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
Robinson
Clifford R
CR
Robinson
Anne Skaja
AS
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000737:chemistry; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014508:Urea; D001617:beta-Glucosidase; D019791:Guanidine