tailieunhanh - Báo cáo khoa học: Identification of three phases in Onconase refolding

Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJÆmol )1 in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase, refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases (fast, medium, and slow), with rate constants differing by a factor of about 10 each. | ễFEBS Journal Identification of three phases in Onconase refolding Cindy Schulenburg1 z Maria M. Martinez-Senac1 z Christian Low2 Ralph Golbik1 Renate Ulbrich-Hofmann1 and Ulrich Arnold1 1 Institute of Biochemistry and Biotechnology Martin-Luther University Halle-Wittenberg Halle Germany 2 Institute of Physics Martin-Luther University Halle-Wittenberg Halle Germany Keywords intermediate Onconase proline isomerization protein folding reactivation Correspondence U. Arnold Martin-Luther University Halle-Wittenberg Institute of Biochemistry and Biotechnology Kurt-Mothes Str. 3 06120 Halle Germany Fax 49 345 5527303 Tel. 49 345 5524865 E-mail . de These authors contributed equally to this work Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJ-mol-1 in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases fast medium and slow with rate constants differing by a factor of about 10 each. None of the phases could be accelerated by peptidyl-prolyl cistrans isomerases pointing to the absence of kinetic phases that are limited by Pro isomerization. The detailed analysis by various probes indicates that the burial of the N-terminal Trp3 which is associated with the restoration of the active site occurs in the slow phase . in the last step of refolding. Evidently in contrast to the folding of RNase A there is no catalytically active native-like intermediate in the folding of Onconase. Received 23 July 2007 revised 30 August 2007 accepted 12 September 2007 doi The smallest member of the RNase A superfamily is Onconase ONC EC Alfacell Corp. Somerset NJ also known as P-30 protein or ranpirn-ase an RNase from the Northern leopard frog Rana pipiens 1 . Despite .