Pedro L. Mateo

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase

The oxidative refolding of hen lysozyme has been studied by a variety of time‐resolved biophysical methods in conjunction with analysis of folding intermediates using reverse‐phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four‐disulfide‐containing fully native protein via rearrangement of a highly native‐like, kinetically trapped intermediate, des‐[76–94], although a significant population (∼30%) appears to fold more quickly via other three‐disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des‐[76–94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.

Comparative thermodynamic study of pepsinogen and pepsin structure

Abstract Pepsinogen, pepsin and its C-terminal fragment have been studied thermodynamically in solution by a scanning microcalorimetric method at various pH and salt content values. It has been shown that: 1. (1) thermal denaturation of pepsinogen is a highly reversible process that takes place within a narrow temperature range depending upon the prevalent conditions, but under no condition does it correspond to a two-state transition. This process can be approximated by two quasi-independent transitions, indicating that the pepsinogen molecule consists of two slightly interacting, co-operative structural blocks of different size. 2. (2) Thermal denaturation of pepsin is a complex process that proceeds in two distinct stages occurring at different temperatures, only the second being completely reversible. These stages correspond to separate meltings of two independent parts of the molecule, these being the N-terminal lobe and the more stable C-terminal lobe. Neither of these stages represents a two-state transition. Analysis of these transitions shows that both parts of the pepsin molecule consist of two quasi-independent, co-operative units. 3. (3) All four co-operative units of pepsin have a compact structure with a welldeveloped hydrophobic core, and therefore these units should be regarded as structural domains of the molecule. Consequently, each lobe of the pepsin molecule represents a structural block consisting of two domains. In the presence of pepstatin, the two domains in the N terminal lobe co-operate to form a single system. 4. (4) In pepsinogen, the above-mentioned blocks are much less independent than the co operative units in pepsin, and it is likely that each of them consists of two merged domains. Therefore, release of the 44-residue N-terminal polypeptide upon activation of pepsinogen leads to a loosening of the domain structure and to an increase of interdomain motility of the molecule.

A thermodynamic analysis of a family of small globular proteins: SH3 domains

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

A Thermodynamic and Kinetic Analysis of the Folding Pathway of an SH3 Domain Entropically Stabilised by a Redesigned Hydrophobic Core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammonds postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

AS-48: a circular protein with an extremely stable globular structure

The unfolding thermodynamics of the circular enterocin protein AS‐48, produced by Enterococcus faecalis, has been characterized by differential scanning calorimetry. The native structure of the 70‐residue protein is extremely thermally stable. Thus, at pH 2.5 and low ionic strength thermal denaturation occurs under equilibrium at 102°C, while the unfolded state irreversibly aggregates at neutral and alkaline pH. Calorimetric data analysis shows that the specific enthalpy change upon unfolding is unusually small and the heat capacity change is quite normal for a protein of this size, whereas the Gibbs energy change at 25°C is relatively high. At least part of this high stability might be put down to entropic constraints induced by the circular organization of the polypeptide chain.

Thermal transitions in the purple membrane from Halobacterium halobium

The application of a successive annealing procedure to the scanning calorimetric endotherm of the purple membrane from Halobacterium halobium in phosphate buffer, pH 7.5, leads to five thermal transitions beneath the overall endotherm. Circular dichroism and fluorescence experiments have also been carried out with the native membrane heated at the same scan rate as in calorimetric runs (1°C/min) as well as with previously heated membrane samples. These results, together with others from the literature, have been used to suggest a preliminary explanation of the five thermal transitions.

Pepsinogen denaturation is not a two-state transition

1. introduction The knowledge of the mode of the denaturation reaction of proteins is important not only to under- stand their mechanism of folding, but also to obtain infomlation on their structural organization. From this point of view pepsinogen is one of the most inter- esting objects, since it is sufficiently large (M, 40 000) and its denaturation is highly reversible, which is a rare situation among proteins. It has been proposed in kinetic studies of pepsinogen denaturation that this process is a two-state transition [1,2], i.e., that the structure of this protein represents a single cooperative system. On this basis the thermodynamic parameters of stabilization of the native structure of pepsinogen have been obtained from equilibrium measurements of denaturation [3]. Here, we present the results of a calorimetric study of the thermodynamics of pepsinogen denaturation which definitely show that this process is not a two- state transition. It is highly probable that a pepsino- gen molecule consists of two quasi-independent and thermodynamically rather identical cooperative blocks. The tllermodynamic similarity of these two blocks seems to be the main reason that their existence has not been revealed by kinetic studies. 2. Materials and methods Grade 1 swine stomach pepsinogen, chromatograph- ically free of pepsin activity, was purchased from Sigma (lot 98C-0451), as a lyophilized powder, and used without further purification. Protein solutions were prepared by dissolving the lyophilized powder *

Heat and cold denaturation of β-lactoglobulin B

The thermal denaturation of bovine β‐lactoglobulin B was investigated by high‐sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 2OmM phosphate buffer. The process was found to be a reversible, two‐state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low‐temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of β‐lactoglobulin B.

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.

Crystallographic structure of the SH3 domain of the human c‐Yes tyrosine kinase: Loop flexibility and amyloid aggregation

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline‐rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high‐resolution crystal structure of the SH3 domain of the c‐Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n‐Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.