Untangling the folding mechanism of the 52-knotted protein UCH-L3

During the last two decades, an enormous amount of information has been obtained on how proteins fold into their distinctive, 3D structures. Many smaller pro-teins or protein domains, usually < 100 residues in length, have been thoroughly studied in terms of their folding pathways [1], allowing pieces of the folding puzzle to be solved and mechanisms to be proposed. By contrast, the folding of larger proteins and oligo-meric protein complexes has not been investigated to the same extent. There is an even greater dearth of knowledge on the folding mechanisms of polypeptides belonging to the group of proteins possessing deep topological crossings in their polypeptide chains known as ‘knots ’ [2]. These so-called knotted proteins ( 273 have been discovered to date) have different structures and biological functions, but share an unu-sual trait in which the polypeptide chain is threaded through a loop formed by a different part of the poly-peptide chain to form deep knot-like structures [2,3]. Many of these proteins are found in prokaryotes, for example, the simple trefoil-knotted methyltransferases, YibK and YbeA. Folding studies on these knotted proteins have begun to reveal aspects of the threading and knotting processes that appear to be required for the protein to achieve its active, native structure [4–12]. Recently, however, even more complicated knotted proteins have been discovered in higher eukaryotes such as plants and humans [2,3]. This is Keywords folding kinetics; hyperfluorescent intermediate(s); knotted proteins; protein folding; ubiquitin C-terminal hydrolas