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.

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic schemewhere k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.Keywords: horeseradish peroxidase; differential scanning calorimetry; intrinsic fluorescence; circular dichroism; irreversible denaturation.Horseradish peroxidase (HRP) belongs to the superfamily of the heme-containing plant peroxidases (EC 1.11.1.7), which has been divided into three classes [1], supported in the first instance by comparison of amino-acid sequence data and confirmed by more recent data on crystal structures [2]. Plant peroxidases, including HRP, comprise class III of the superfamily. Although the function of peroxidases is often seen primarily in terms of the conversion of H 2 O 2 to H 2 O, this should not be allowed to mask their wider participation in other reactions, many of which are biologically significant. Despite the enormous interest in peroxidases owing to their broad practical applications in biotechnology, the data concerning their structural stability are sparse. Although several publications have addressed the thermal stability of peroxidases [3±8], to date the mechanism of the process of thermal denaturation remains unclear. It is known that the biological functions of proteins depend on the correct folding of their native structure and that loss of this folded structure leads to an unfolded, inactive state. Consequently, the study of protein stability is important both from the academic and applied points of view.Factors affecting conformational stability have been studied most intensively in proteins under reversible conditions [9±15]. Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturat...

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.

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