Complex kinetics and residual structure in the thermal unfolding of yeast triosephosphate isomerase.

BACKGROUND: Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature. RESULTS: Thermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, ß-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a ß-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility. CONCLUSIONS: The slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. ß-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.

Antioxidant capacity and structural changes of human serum albumin from patients in advanced stages of diabetic nephropathy and the effect of the dialysis.

Changes in the antioxidant capacity of albumin and alterations of the albumin structural conformation were examined in patients in advanced stages of diabetes nephropathy. Human serum albumin was purified from diabetic patients in pre-dialysis (glomerular filtration rate [GFR] between 15 and 29 ml min(-1) 1.73 m(-2)) and those in dialysis (GFR ≤ 15 ml min(-1) 1.73 m(-2)) and then compared with albumin from patients with a normal GFR (>90 ml min(-1) m(-2)). We evaluated the antioxidant capacity of albumin using an enhanced chemiluminescence-based assay and thiol group content, and the structural changes were evaluated by circular dichroism and fluorescence spectroscopy. The antioxidant capacity and thiol content of albumin from patients in advanced stages of diabetic nephropathy were markedly reduced. The circular dichroism spectra showed a mean albumin α-helix content reduction from 44 to 37 % and from 44 to 30 % between the control group and pre-dialysis and dialysis patients, respectively. Additionally, the fluorescence intensity was reduced by 4.2 and 13 % for the groups 4 and 5, respectively, in relation with the control. These data provide evidence for the partial denaturation of albumin and exacerbated oxidative stress among patients in advanced stages of diabetes nephropathy before and even after dialysis.

Additive effect of single amino acid replacements on the kinetic stability of ß-glucosidase B.

Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of ß-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG ( N→TS )) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C ( ß ) s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.