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Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP β-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation. Portuguese national funds from FCT - Foundation for Science and Technology UID/Multi/04326/2019;Portugal 2020, CRESC Algarve 2020, Lisboa 2020 European Union (EU) 17653 info:eu-repo/semantics/publishedVersion

Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.

Resumo: A varicela é uma doença infectocontagiosa comum na infância, ocorrendo pouco mais de 2% dos casos em adultos. Desde a década de 80 que a sua incidência nos adultos tem vindo a aumentar, dos quais apenas 7% são seronegativos1. A pneumonia a Varicella zoster, se bem que rara, constitui a complicação mais grave e mais frequente no adulto.Os autores apresentam um caso clínico ilustrativo de pneumonia a Varicella zoster num adulto fumador e imunocompetente e fazem uma breve revisão teórica sobre o tema.Rev Port Pneumol 2010; XVI (3): 493-505 Abstract: Varicella (chickenpox) is a common contagious infection of childhood, with fewer than 2% of the cases occurring in adults. Since the early 1980s the incidence of chickenpox in adults has been increasing and only 7% of them are seronegative for Varicella zoster antibodies. Pneumonia, although rare, is the most common and serious complication of chickenpox infection in adults.The authors present an illustrative case of varicella pneumonia in an immunocompetent adult with smoking habits and make a brief thematic review.Rev Port Pneumol 2010; XVI (3): 493-505 Palavras-chave: Varicela, pneumonia, ARDS, Key-words: Chickenpox, pneumonia, ARDS

The stability of the Aquifex aeolicus multicopper oxidase (McoA) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. The enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 °C. Chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depletion occur before the unfolding of the tertiary structure and McoA is highly prone to aggregate. Indeed, unfolding kinetics measured with the stopped-flow technique quantified the stabilizing effect of copper on McoA (1.5 kcal/mol) and revealed quite an uncommon observation further confirmed by light scattering and gel filtration chromatography: McoA aggregates in the presence of guanidinium hydrochloride, i.e., under unfolding conditions. The aggregation process results from the accumulation of a quasi-native state of McoA that binds to ANS and is the main determinant of the stability curve of McoA. Kinetic partitioning between aggregation and unfolding leads to a very low heat capacity change and determines a flat dependence of stability on temperature.

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.

Horseradish peroxidase A2 is the most abundant anionic peroxidase in horseradish roots. Steady-state fluorescence, circular dichroism and differential scanning calorimetry were used to study the thermal stability of HRPA2 at pH values of 4, 7 and 10. Changes in the intrinsic protein probes follow the increasing order: tryptophan fluorescence, secondary structure, and heme group environment at pH 4 and 7 showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. At pH 10, the heme environment is the first structural region to suffer conformational alterations concurrently with local tryptophan environmental changes. The denaturation process was analyzed by the classic scheme, N ↔ U → D used to fit thermal denaturation, followed by far-UV CD ellipticity. This model described well the experimental Tm values, their dependence on the scan rate and the percentages of irreversibility after thermal denaturation. Thermal denaturation is partially reversible at pH 10 and 7 while a 100% irreversible process was observed at pH 4. Despite the accurate fit of data by this pathway intermediate states must populate the equilibrium between N and U as changes in the intrinsic protein probes were not coincident.

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.

The effect of trehalose on folding and stability of the small ribosomal protein S6 was studied. Non-disruptive point mutations distributed along the protein structure were analyzed to characterize the stabilizing effect of trehalose and map the folding pathway of S6. On average, the stability of the wild-type and S6 mutants increases by 3 kcal/mol M trehalose. Despite the non-specific thermodynamic stabilization mechanism, trehalose particularly stabilizes the less destabilized mutants. Folding/unfolding kinetics shows clearly that trehalose induces the collapse of the unfolded state to an off-pathway intermediate with non-native diffuse contacts. This state is similar to the collapsed state induced by high concentrations of stabilizing salts, as previously reported. Although it leads to the accumulation of this off-pathway intermediate, trehalose does not change the compactness of the transition state ensemble. Furthermore, the productive folding pathway of S6 is not affected by trehalose as shown by a Phi-value analysis. The unfolded state ensemble of S6 should be more compact in the presence of trehalose and therefore destabilized due to decreased conformational entropy. Increased compaction of the unfolded state ensemble might also occur for more stable mutants of S6, thus explaining the synergistic effect of trehalose and point mutations on protein stabilization.

Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca2+-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, ICa2+, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of ICa2+ increases exponentially with increasing calcium concentration. The ICa2+ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state.

The stability of a recombinant cutinase from the fungus Fusarium solani was evaluated in aqueous media and in reverse micelles. Thermal unfolding in aqueous solution is a two-state process at the pH values tested and trehalose increased the temperature at the mid-point of the unfolding transitions. Irreversible inactivation is a first-order process at pH 9.2, but two inactivation phases were resolved at pH 4.5. Trehalose did not change the irreversible inactivation pathway but increased the kinetics of the irreversible inactivation step. Unfolding of cutinase induced by guanidine hydrochloride was more complex, showing a stable intermediate, molten globule in character, within the transition region. Trehalose did not change the three-state nature of the unfolding process. Encapsulation of cutinase in AOT reverse micelles induced unfolding at room temperature due to an enzyme location at the micellar interface. The presence of 1-hexanol as co-surfactant delayed or even prevented the unfolding of cutinase by promoting the establishment of a new equilibrium in the system. Cutinase is encapsulated in a 10-fold larger AOT/hexanol reverse micelle built up by the fusion of empty reverse micelles. When tested in a membrane reactor in the presence of 1-hexanol, an operational half-life of 674 days was achieved.

This work describes a methodology to monitor protein unfolding by using the well known changes in tyrosine absorbance with the ionization of the side chain phenol group. It can be applied to proteins that are functionally active at pH values higher than 9.0 where the current UV differential spectroscopy technique can not be used. The simplicity and facility of the proposed methodology (only two absorbance measurements have to be acquired) can make it very useful namely for technological applications. Thermal unfolding of cutinase and alpha-chymotrypsin were followed using this methodology and the thermodynamic stability data were obtained assuming a two-state mechanism. The transition from the folded to the unfolded state was further confirmed by fluorescence maxima for both proteins proving the validity of the methodology based on UV measurements.