Polyamines are positively charged small molecules ubiquitously existing in all living organisms, and they are considered as one kind of the most ancient cellular components. The most common polyamines are spermidine, spermine, and their precursor putrescine generated from ornithine. Polyamines play critical roles in cells by stabilizing chromatin structure, regulating DNA replication, modulating gene expression, etc., and they also affect the structure and function of proteins. A few studies have investigated the impact of polyamines on protein structure and function previously, but no reports have focused on a protein-based biological module with a dedicated function. In this report, we investigated the impact of polyamines (putrescine, spermidine, and spermine) on the cyanobacterial KaiABC circadian oscillator. Using an established in vitro reconstitution system, we noticed that polyamines could disrupt the robustness of the KaiABC oscillator by inducing the denaturation of the Kai proteins (KaiA, KaiB, and KaiC). Further experiments showed that the denaturation was likely due to the induced change of the thermal stability of the clock proteins. Our study revealed an intriguing role of polyamines as a component in complex cellular environments and would be of great importance for elucidating the biological function of polyamines in future.

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.

Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now.

We have combined spectroscopy, chromatography, calorimetry, and small-angle X-ray scattering (SAXS) to provide a comprehensive structural and stoichiometric description of the sodium dodecyl sulfate (SDS)-induced denaturation of the 86-residue alpha-helical bovine acyl-coenzyme-A-binding protein (ACBP). Denaturation is a multistep process. Initial weak binding of 1-3 SDS molecules per protein molecule below 1.3 mM does not perturb the tertiary structure. Subsequent binding of approximately 13 SDS molecules per ACBP molecule leads to the formation of SDS aggregates on the protein and changes in both tertiary and secondary structures. SAXS data show that, at this stage, a decorated micelle links two ACBP molecules together, leaving about half of the polypeptide chain as a disordered region protruding into the solvent. Further titration with SDS leads to the additional uptake of 26 SDS molecules, which, according to SAXS, forms a larger decorated micelle bound to a single ACBP molecule. At the critical micelle concentration, we conclude from reduced mobility and increased fluorescence anisotropy that each ACBP molecule becomes associated with more than one micelle. At this point, 56-60 SDS molecules are bound per ACBP molecule. Our data provide key structural insights into decorated micelle complexes with proteins, revealing a remarkable diversity in the different conformations they can stabilize. The data highlight that a minimum decorated micelle size, which may be a key driving force for intermolecular protein association, exists. This may also provide a structural basis for the known ability of submicellar surfactant concentrations to induce protein aggregation and fibrillation.

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO2 (4-8 nm) and CuxO (1-2 nm or less). Their extraordinarily small size leads to high dispersity and good optical transparency, besides large active surface area. Those photocatalysts can be applied to white and translucent latex paints. Although Cu2O clusters involved in the paint coating undergo gradual aerobic oxidation in the dark, the oxidized clusters are re-reduced under > 380 nm light. The paint coating inactivated the original and alpha variant of novel coronavirus under irradiation with fluorescent light for 3 h. The photocatalysts greatly suppressed binding ability of the receptor binding domain (RBD) of coronavirus (the original, alpha and delta variants) spike protein to the receptor of human cells. The coating also exhibited antivirus effects on influenza A virus, feline calicivirus, bacteriophage Qβ and bacteriophage M13. The photocatalysts would be applied to practical coatings and lower the risk of coronavirus infection via solid surfaces.

In this study, the influence of defined extrusion-like treatment conditions on the denaturation behavior and kinetics of single- and multi-component protein model systems at a protein concentration of 70% (w/w) was investigated. α-Lactalbumin (αLA) and β-Lactoglobulin (βLG), and whey protein isolate (WPI) were selected as single- and multi-component protein model systems, respectively. To apply defined extrusion-like conditions, treatment temperatures in the range of 60 and 100 °C, shear rates from 0.06 to 50 s⁻1, and treatment times up to 90 s were chosen. While an aggregation onset temperature was determined at approximately 73 °C for WPI systems at a shear rate of 0.06 s⁻1, two significantly different onset temperatures were determined when the shear rate was increased to 25 and 50 s⁻1. These two different onset temperatures could be related to the main fractions present in whey protein (βLG and αLA), suggesting shear-induced phase separation. Application of additional mechanical treatment resulted in an increase in reaction rates for all the investigated systems. Denaturation was found to follow 2.262 and 1.865 order kinetics for αLA and WPI, respectively. The reaction order of WPI might have resulted from a combination of a lower reaction order in the unsheared system (i.e., fractional first order) and higher reaction order for sheared systems, probably due to phase separation, leading to isolated behavior of each fraction at the local level (i.e., fractional second order).

A method that can pinpoint control DNA denaturation is reported. In the single molecule experiment using spFRET, DNA adhered on a quartz surface is acted upon by both a weak laser field force and a fast temporal mechanical force. The experiment showed that increasing strengths of laser power result in increasing percentage of denatured DNA; different mechanical forces produce different numbers of DNA opening. Besides the method's simplicity and convenience for DNA melting, its crucial advantage and potential application is the ability to denature DNA at specified locations, i.e., a weak laser and a fast temporal mechanical force can be used in pinpoint denaturation of short DNA.

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as 'Trishanku' unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native 'Trishanku' refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.

Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0-1 mM SL), it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and non-ionic or zwitterionic surfactants. Inclusion of diacetylated lactonic sophorolipid (lactSL) as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent and pharmaceutical industry.

Peptides produced by the proteasome have been proposed to function as signaling molecules that regulate a number of biological processes. In the current study, we used quantitative peptidomics to test whether conditions that affect protein stability, synthesis, or turnover cause changes in the levels of peptides in Human Embryonic Kidney 293T (HEK293T) cells. Mild heat shock (42 °C for 1 h) or treatment with the deubiquitinase inhibitor b-AP15 led to higher levels of ubiquitinated proteins but did not significantly increase the levels of intracellular peptides. Treatment with cycloheximide, an inhibitor of protein translation, did not substantially alter the levels of intracellular peptides identified herein. Cells treated with a combination of epoxomicin and bortezomib showed large increases in the levels of most peptides, relative to the levels in cells treated with either compound alone. Taken together with previous studies, these results support a mechanism in which the proteasome cleaves proteins into peptides that are readily detected in our assays (i.e., 6-37 amino acids) and then further degrades many of these peptides into smaller fragments.

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing-thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.

Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+ , and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.

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.