David G. Pina

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. 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 necessarily limit folding in vitro, however, it does seem to require a complex folding mechanism which includes the formation of several distinct intermediate species.

Thermostability of cardosin A from Cynara cardunculus L.

The structural stability of cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculusL., has been investigated by high-sensitivity differential scanning calorimetry, intrinsic fluorescence and circular dichroism spectroscopy, and enzymatic activity assays. Even though the thermal denaturation of cardosin A is partially irreversible, valid thermodynamic data can be obtained within a wide pH region. Also, although cardosin A is a heterodimeric enzyme its thermal denaturation occurs without simultaneous dissociation to unfolded monomers. Moreover, in the 3–7 pH region the excess heat capacity can be deconvoluted into two components corresponding to two elementary two-state transitions, suggesting that the two polypeptide chains of cardosin A unfold independently. Detailed thermodynamic and structural investigations of cardosin A at pH = 5.0, at which value the enzyme demonstrates maximum stability and enzymatic activity, revealed that after thermal denaturation the polypeptide chains of this protein retain most of their secondary structure motifs and are not completely hydrated.

Correlation between Shiga toxin B-subunit stability and antigen crosspresentation: A mutational analysis

The homopentameric B‐subunit of Shiga toxin (STxB) is used as a tool to deliver antigenic peptides and proteins to the cytosolic compartment of dendritic cells (DCs). In this study, a series of interface mutants of STxB has been constructed. All mutants retained their overall conformation, while a loss in thermal stability was observed. This effect was even more pronounced in trifluoroethanol solutions that mimic the membrane environment. Despite this, all mutants were equally efficient at delivering a model antigenic protein into the MHC class I‐restricted antigen presentation pathway of mouse DCs, suggesting that the structural stability of STxB is not a key factor in the membrane translocation process.