tailieunhanh - Báo cáo khoa học: Untangling the folding mechanism of the 52-knotted protein UCH-L3

Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein-folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C-terminal hydrolase UCH-L3, which is an integral part of the ubiquitin–proteasome system. | ỊFEBS Journal Untangling the folding mechanism of the 52-knotted protein UCH-L3 Fredrik I. Andersson David G. Pina Anna L. Mallam Georg Blaser and Sophie E. Jackson University ChemicalLaboratory Cambridge UK Keywords folding kinetics hyperfluorescent intermediate s knotted proteins protein folding ubiquitin C-terminalhydrolase Correspondence S. Jackson University ChemicalLaboratory Lensfield Road Cambridge CB2 1EW UK Fax 44 1223 336362 Tel 44 1223 762011 E-mail sej13@ Received 9 December 2008 revised 27 February 2009 accepted 3 March 2009 doi Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein-folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C-terminal hydrolase UCH-L3 which is an integral part of the ubiquitin-proteasome system. The structure of UCH-L3 contains five distinct crossings of its polypeptide chain and it adopts a 52-knotted topology making it a fascinating target for folding studies. Here we provide the first in depth characterization of the stability and folding of UCH-L3. We show that the protein can unfold and refold reversibly in vitro without the assistance of molecular chaperones demonstrating that all the information necessary for the protein to find its knotted native structure is encoded in the amino acid sequence just as with any other globular protein and that the protein does not enter into any deep kinetic traps. Under equilibrium conditions the unfolding of UCH-L3 appears to be two-state however multiphasic folding and unfolding kinetics are observed and the data are consistent with a folding pathway in which two hyperfluorescent intermediates are formed. In addition a very slow phase in the folding kinetics is shown to be limited by proline-isomerization events. Overall the data suggest that a knotted topology even in its most complex form does not .