Low concentration of guanidine hydrochloride induces the formation of an aggregation-prone state in alpha-urease.
Canavalia ensiformis (jack bean) alpha-urease is a hexameric protein characterized by a complex denaturation mechanism. In previous papers, we have shown that a hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANSA) binding conformer could be populated in a moderate concentration of denaturant. This state was obtained under conditions that had no detectable impact on its tertiary structure, as indicated by fluorescence measurements. In the present study, we further characterized this ANSA-binding state in an attempt to understand urease behavior. Evidence presented here shows that the presence of ANSA was not required for the generation of the conformer and that its affinity for ANSA came from an increase in hydrophobicity leading to aggregation. Circular dichroism investigation of urease revealed that it had periodical secondary structure content similar to Klebsiella aerogenes urease (secondary structures calculated on the basis of crystallographic data). The impact of 0.9 M guanidine hydrochloride (GuHCl) on soluble urease secondary structures was minimal but is compatible with a slight increase in beta-sheet structures. Such modification may indicates that aggregation involves amyloid-like fibril formation. Electron microscopy analysis of urease in the absence of GuHCl revealed the presence of urease hexamers (round shape 13 nm in diameter). These particles disappeared in the presence of moderate denaturant concentration owing to the formation of aggregates and fibril-like structures. The fibrils obtained in 1.5 M GuHCl had an average diameter of 6.5 nm, suggesting that urease hexamers dissociated into smaller oligomeric forms when forming such fibrils.
McDuff
F-O
FO
Groupe de Recherche en Energie et Information Biomoléculaire, Dépatrement de Chimie-Biologie, Université du Québec à Trois-Rivières, QC, Canada.
Doucet
A
A
Beauregard
M
M
D000817:Anilino Naphthalenesulfonates; D032449:Canavalia / Q000201:enzymology*; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D019791:Guanidine; D008027:Light; D008854:Microscopy, Electron; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D014510:Urease / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014510:Urease; D019791:Guanidine