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Protein denaturation is under intensive research,since it leads to neurological disorders of severe consequences.Avoiding denaturation and stabilizing the proteins in their native state is of great importance,especially when proteins are used as drug molecules or vaccines.It is preferred to add pharmaceutical excipients in protein formulations to avoid denaturation and thereby stabilize them.The present study aimed at using bile salts(BSs),a group of well-known drug delivery systems,for stabilization of proteins.Bovine serum albumin(BSA)was taken as the model protein,whose association with two BSs,namely sodium cholate(NaC)and sodium deoxycholate(NaDC),was studied.Denaturation studies on the preformed BSA-BS systems were carried out under chemical and physical denaturation conditions.Urea was used as the chemical denaturant and BSA-BS systems were subjected to various temperature conditions to understand the thermal(physical)denaturation.With the denaturation conditions prescribed here,the data obtained is informative on the association of BSA-BS systems to be hydrophobic and this effect of hydrophobicity plays an important role in stabilizing the serum albumin in its native state under both chemical and thermal denaturation.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we “reverse engineered” PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.

The Selectivity Filter (SF) in tetrameric K + channels, has a highly conserved sequence, TVGYG, at the extracellular entry to the channel pore region. There, the backbone carbonyl oxygens from the SF residues, create a stack of K + binding sites where dehydrated K + binds to induce a conductive conformation of the SF. This increases intersubunit interactions and confers a higher stability to the channel against thermal denaturation. Indeed, the fit of dehydrated K + to its binding sites is fundamental to define K + selectivity, an important feature of these channels. Nonetheless, the SF conformation can be modified by different effector molecules. Such conformational plasticity opposes selectivity, as the SF departs from the “induced-fit” conformation required for K + recognition. Here we studied the KirBac1.1 channel, a prokaryotic analog of inwardly rectifying K + channels, confronted to permeant (K + ) and non-permeant (Na + ) cations. This channel is pH-dependent and transits from the open state at neutral pH to the closed state at acidic pH. KirBac1.1 has the orthodox TVGYG sequence at the SF and thus, its behavior should resemble that of K + -selective channels. However, we found that when at neutral pH, KirBac1.1 is only partly K + selective and permeates this ion causing the characteristic “induced-fit” phenomenon in the SF conformation. However, it also conducts Na + with a mechanism of ion passage reminiscent of Na + channels, i.e., through a wide-open pore, without increasing intersubunit interactions within the tetrameric channel. Conversely, when at acidic pH, the channel completely loses selectivity and conducts both K + and Na + similarly, increasing intersubunit interactions through an apparent “induced-fit”-like mechanism for the two ions. These observations underline that KirBac1.1 SF is able to adopt different conformations leading to changes in selectivity and in the mechanism of ion passage.

To investigate the mechanism of the texture formed by protein thermal denaturation, the profile and formation of texture and thermal denaturation of protein were evaluated using texture profile analysis (TPA) and transmission electron microscopy (TEM) combined with differential scanning calorimeter (DSC). Results indicated that the surface temperature of Beijing roast duck increased from 23.9 to 174.4 °C, while the center temperature rose from 20.6 to 99.3 °C during roasting. Shear force decreased significantly during the first 20 min, and the texture profile largely changed at 20 and 40 min. Firstly, Band I was broken and twisted, Band A was overstruck, and Z-line was diffused and finally disappeared, resulting in a blurred myofibril structure. The sarcomere considerably contracted within 30 min. Secondly, the main myofibrillar proteins were denatured at 20 and 40 min, respectively. The formation of hydrophobic interactions and the reduction of ionic bonds were observed. Thirdly, roasting induced protein thermal denaturation, which was correlated with interprotein forces, texture profile, and the shear force. Muscle fibers were damaged and shrunken, accompanied by the formation of hydrophobic interactions and the reduction of ionic bonds. The turning points were at 20 and 40 min, and the main proteins were denatured, leading to the formation of tenderness of Beijing roast duck.

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T m), at pH 2.0-8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T m values of both holo bLF and holo cLF were reduced showing T m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.

Temperature-induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized. We developed a structural proteomic strategy to measure protein thermostability on a proteome-wide scale and with domain-level resolution. We applied it to , , , and human cells, yielding thermostability data for more than 8000 proteins. Our results (i) indicate that temperature-induced cellular collapse is due to the loss of a subset of proteins with key functions, (ii) shed light on the evolutionary conservation of protein and domain stability, and (iii) suggest that natively disordered proteins in a cell are less prevalent than predicted and (iv) that highly expressed proteins are stable because they are designed to tolerate translational errors that would lead to the accumulation of toxic misfolded species.

Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation).

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at −80 °C consumes as much energy per day as a small household 1 . Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose 2 , sucrose or polymers 3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze–thaw 4 , leading to a very complex and expensive supply chain. Other ideas involve ensilication 5 and chemical modification of proteins 6 . In short, protein stabilization is a challenge with no universal solution 7 , 8 . Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide. A stiff hydrogel gel is presented that encapsulates and stabilizes proteins without additives or excipients and uses mechanical strain to release them, offering low-cost and versatile delivery of therapies.

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

Post-transcriptional modifications have critical roles in tRNA stability and function 1 – 4 . In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures 5 , 6 . Here we identified 2′-phosphouridine (U p ) at position 47 of tRNAs from thermophilic archaea. U p 47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by U p 47. The 2′-phosphate of U p 47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for U p 47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates U p 47 in vitro and in vivo. Taken together, our findings show that U p 47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions. Reversible internal RNA phosphrylation contributes to thermal stability and nuclease resistance of tRNA, and cellular thermotolerance of hyperthermophiles.