Molten globule-like transition state of protein barnase measured with calorimetric force spectroscopy.

SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.

Influence of divalent metal cations on α-lactalbumin fibril formation.

The effect of binding of divalent metal cations (Ca2+, Cu2+, Mg2+, Mn2+, Zn2+) on the kinetics of fibril formation of bovine α-lactalbumin at acidic conditions is considered. The kinetic parameters of the process were determined using a thioflavin T fluorescence assay. The DSC thermograms of bovine α-lactalbumin in the presence and absence of cations were recorded. The duration of the lag period correlates with the changes in the thermal stability of the molten globule of the protein in the presence of cations. The final thioflavin T fluorescence intensity after formation of the mature fibrils decreases under the influence of calcium ions which strongly bind to the monomeric protein, and increases in solutions containing copper and especially zinc. These ions seem to accelerate secondary nucleation processes and change the fibril morphology, which was confirmed by atomic force microscopy imaging.

Boolean Logic Gate Operation in Bacteriorhodopsin of Purple Membrane Based on a Molten Globule-like State.

Bacteriorhodopsin (bR) of purple membrane (PM) has increasing technical interests, particularly in photonic devices and bioelectronics. The present work has concerned with monitoring the temperature dependence of passive electric responses in-plane and out-of-plane of the membranes. Based on thermal properties observed orthogonally here for PM, a high-temperature intermediate of bR has been suggested to populate at around 60 °C, which may be ascribed to a molten globule-like state. This intermediate has been found to be enclosed between two reversible thermal transitions for PM. Large-scale turnover in the energy of activation, for these two thermal transitions, occurs steeply at such state at 60 °C, above which does bR reverse the sign of dielectric anisotropy (i.e. crossover) provided the operating frequency should be above the crossover frequency, at which the reversal occurs. No such crossover was found to occur below the crossover frequency, even above the crossover temperature (i.e. 60 °C). Likewise, no such crossover was found to occur below the crossover temperature, even above the crossover frequency. Relying on this reasoning, a logic gate operation may be declared implicating bR for bioelectronics and sense technological relevance. In addition, the results specify "dual frequency" as well as "dual temperature" characteristics to bacteriorhodopsin.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Do "Newly Born" orphan proteins resemble "Never Born" proteins? A study using three deep learning algorithms.

"Newly Born" proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by the expression of novel open reading frames, and appear throughout evolution. We were curious if three recently developed programs for predicting protein structures, namely, AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such "Newly Born" proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called "Never Born" proteins. The programs were used to compare the structures of two sets of "Never Born" proteins that had been expressed-Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, although the models generated were scored as being of low quality, they nevertheless revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms, in agreement with the experimental data, whereas the members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins, again consistent with the experimental data. These predicted differences were shown to be statistically significant by comparing their accessible surface areas. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Surprisingly, only for the protein which did not have a novel fold, and was taxonomically restricted, rather than being a true orphan, did all three algorithms predict very similar, high-quality structures, closely resembling the crystal structure. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with only two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member, and to the fact that the Dali server revealed several nonrelated proteins with similar folds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Proteins:3.

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 µs Molecular Dynamics Simulation.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 µs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

The interaction of trehalose and molten globule state soybean 11S globulin and its impact on foaming capacities.

BACKGROUND: Soybean 11S globulin has good functional properties, which are widely used in the field of food. However, natural soybean 11S globulin (N-11S) has low flexibility and is easy to aggregate, impacting its foaming process. Studies have shown that soybean 11S globulin in molten globule state (MG-11S) has better molecular flexibility than N-11S, and trehalose has been shown to improve the properties of proteins. Therefore, this study investigated the interaction mechanism between trehalose and MG-11S, and its impact on rheological and foaming properties of MG-11S. RESULTS: The molecular docking and intrinsic fluorescence results showed that hydrogen bonding was the main interaction force at lower than 0.5 mol L-1 trehalose added. Meanwhile, rheology and foaming showed that the MG-11S-trehalose complexes had better viscoelasticity, foaming ability (66.67-86.67%) and foaming stability (75.00-89.29%) than N-11S (16.67% foaming ability and 40.00% foaming stability); however, when the trehalose was higher than 0.5 mol L-1 , molecular crowding occurred and H-bonds were weakened, resulting in reduction of foaming capacities. Microstructure determination showed that trehalose attached to the surface of foam membrane; meanwhile, the foaming structure of the complex with 0.5 mol L-1 trehalose had a thicker liquid film with decreased drainage rate, less agglomeration and disproportionation of foam, illustrating the best foaming ability and foaming stability. CONCLUSION: The results suggested that trehalose at different concentrations can interact with MG-11S through different mechanisms, and improve the foaming capacity of MS-11S. This provided a reference for the application of MS-11S in foaming food. © 2022 Society of Chemical Industry.

The C-terminal tail extension of myosin 16 acts as a molten globule, including intrinsically disordered regions, and interacts with the N-terminal ankyrin.

The lesser-known unconventional myosin 16 protein is essential in proper neuronal functioning and has been implicated in cell cycle regulation. Its longer Myo16b isoform contains a C-terminal tail extension (Myo16Tail), which has been shown to play a role in the neuronal phosphoinositide 3-kinase signaling pathway. Myo16Tail mediates the actin cytoskeleton remodeling, downregulates the actin dynamics at the postsynaptic site of dendritic spines, and is involved in the organization of the presynaptic axon terminals. However, the functional and structural features of this C-terminal tail extension are not well known. Here, we report the purification and biophysical characterization of the Myo16Tail by bioinformatics, fluorescence spectroscopy, and CD. Our results revealed that the Myo16Tail is functionally active and interacts with the N-terminal ankyrin domain of myosin 16, suggesting an intramolecular binding between the C and N termini of Myo16 as an autoregulatory mechanism involving backfolding of the motor domain. In addition, the Myo16Tail possesses high structural flexibility and a solvent-exposed hydrophobic core, indicating the largely unstructured, intrinsically disordered nature of this protein region. Some secondary structure elements were also observed, indicating that the Myo16Tail likely adopts a molten globule-like structure. These structural features imply that the Myo16Tail may function as a flexible display site particularly relevant in post-translational modifications, regulatory functions such as backfolding, and phosphoinositide 3-kinase signaling.

Aberrant environment and PS-binding to calnuc C-terminal tail drives exosomal packaging and its metastatic ability.

The characteristic features of cancer cells are aberrant (acidic) intracellular pH and elevated levels of phosphatidylserine. The primary focus of cancer research is concentrated on the discovery of biomarkers directed towards early diagnosis and therapy. It has been observed that azoxymethane-treated mice demonstrate an increased expression of calnuc (a multi-domain, Ca2+- and DNA-binding protein) in their colon, suggesting it to be a good biomarker of carcinogenesis. We show that culture supernatants from tumor cells have significantly higher amounts of secreted calnuc compared to non-tumor cells, selectively packaged into exosomes. Exosomal calnuc is causal for epithelial-mesenchymal transition and atypical migration in non-tumor cells, which are key events in tumorigenesis and metastasis. In vitro studies reveal a significant affinity for calnuc towards phosphatidylserine, specifically to its C-terminal region, leading to the formation of 'molten globule' conformation. Similar structural changes are observed at acidic pH (pH 4), which demonstrates the role of the acidic microenvironment in causing the molten globule conformation and membrane interaction. On a precise note, we propose that the molten globule structure of calnuc caused by aberrant conditions in cancer cells to be the causative mechanism underlying its exosome-mediated secretion, thereby driving metastasis.

Ephemeral states in protein folding under force captured with a magnetic tweezers design.

Magnetic tape heads are ubiquitously used to read and record on magnetic tapes in technologies as diverse as old VHS tapes, modern hard-drive disks, or magnetic bands on credit cards. Their design highlights the ability to convert electric signals into fluctuations of the magnetic field at very high frequencies, which is essential for the high-density storage demanded nowadays. Here, we twist this conventional use of tape heads to implement one in a magnetic tweezers design, which offers the unique capability of changing the force with a bandwidth of ∼10 kHz. We calibrate our instrument by developing an analytical expression that predicts the magnetic force acting on a superparamagnetic bead based on the Karlqvist approximation of the magnetic field created by a tape head. This theory is validated by measuring the force dependence of protein L unfolding/folding step sizes and the folding properties of the R3 talin domain. We demonstrate the potential of our instrument by carrying out millisecond-long quenches to capture the formation of the ephemeral molten globule state in protein L, which has never been observed before. Our instrument provides the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a piconewton, opening a range of force spectroscopy protocols to study protein dynamics under force.

The Molten Globule State of a Globular Protein in a Cell Is More or Less Frequent Case Rather than an Exception.

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn's hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.

Identification of Two Early Folding Stage Prion Non-Local Contacts Suggested to Serve as Key Steps in Directing the Final Fold to Be Either Native or Pathogenic.

The initial steps of the folding pathway of the C-terminal domain of the murine prion protein mPrP(90-231) are predicted based on the sequential collapse model (SCM). A non-local dominant contact is found to form between the connecting region between helix 1 and ß-sheet 1 and the C-terminal region of helix 3. This non-local contact nucleates the most populated molten globule-like intermediate along the folding pathway. A less stable early non-local contact between segments 120-124 and 179-183, located in the middle of helix 2, promotes the formation of a less populated molten globule-like intermediate. The formation of the dominant non-local contact constitutes an example of the postulated Nature's Shortcut to the prion protein collapse into the native structure. The possible role of the less populated molten globule-like intermediate is explored as the potential initiation point for the folding for three pathogenic mutants (T182A, I214V, and Q211P in mouse prion numbering) of the prion protein.

New Factors Enhancing the Reactivity of Cysteines in Molten Globule-Like Structures.

Protein cysteines often play crucial functional and structural roles, so they are emerging targets to design covalent thiol ligands that are able to modulate enzyme or protein functions. Some of these residues, especially those involved in enzyme mechanisms-including nucleophilic and reductive catalysis and thiol-disulfide exchange-display unusual hyper-reactivity; such a property is expected to result from a low pKa and from a great accessibility to a given reagent. New findings and previous evidence clearly indicate that pKa perturbations can only produce two-four-times increased reactivity at physiological pH values, far from the hundred and even thousand-times kinetic enhancements observed for some protein cysteines. The data from the molten globule-like structures of ribonuclease, lysozyme, bovine serum albumin and chymotrypsinogen identified new speeding agents, i.e., hydrophobic/electrostatic interactions and productive complex formations involving the protein and thiol reagent, which were able to confer exceptional reactivity to structural cysteines which were only intended to form disulfides. This study, for the first time, evaluates quantitatively the different contributions of pKa and other factors to the overall reactivity. These findings may help to clarify the mechanisms that allow a rapid disulfide formation during the oxidative folding of many proteins.

Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer.

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

Determination of the structural ensemble of the molten globule state of a protein by computer simulations.

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.

Conformational diversity in the intrinsically disordered HIV-1 Tat protein induced by zinc and pH.

Human immunodeficiency virus type-1 (HIV-1) transactivator of transcription (Tat) is an intrinsically disordered protein that exerts multiple functions, including activation of HIV-1 replication and induction of T-cell apoptosis and cytokine secretion via zinc binding and cellular uptake by endocytosis. However, the effects of zinc and endosomal low pH on the structure of isolated Tat protein are poorly understood. Here, we purified a monomeric zinc-bound Tat and studied its structure and acid denaturation by circular dichroism, NMR, and small-angle X-ray scattering. We found that at pH 7, the zinc-bound Tat was in a pre-molten globule state; it exhibited largely disordered conformations with residual helices and was slightly more compact than the fully unfolded states that were observed at pH 4 or in the zinc-free form. Moreover, acid-induced unfolding transitions in secondary structure and molecular size occurred at different pH ranges, indicating the presence of an expanded and helical intermediate at pH ∼6. Taken together, the extent of structural disorder in the intrinsically disordered Tat protein is highly sensitive to zinc and pH, suggesting that zinc binding and pH affect Tat structures and thereby control the versatile functions of Tat.

Investigation of structural and saccharide binding transitions of Bauhinia purpurea and Wisteria floribunda lectins.

Two novel medicinally important legume lectins from Bauhinia purpurea (BPL) and Wisteria floribunda (WFL) possessing extended sugar binding site were investigated for functional and conformational transitions using biochemical and biophysical techniques as well as bioinformatical tools. Homology model of BPL was constructed using the Schrodinger suite and docked with N-acetyl galactosamine and T-antigen disaccharide (Galß1-3GalNAcαO-Me). The longer loop D in the structure of WFL compared to that in BPL was found to be responsible for its specificity to LacdiNac (ß-D-GalNAc-[1 → 4]-DGlcNAc) over Galß1-3GalNAc. BPL remained functionally stable up to 40 °C whereas WFL remained stable upto 70 °C indicating the strength of the sugar binding site geometry. Both the lectins showed intense but non-specific secondary structure in the range of 65-90 °C. WFL showed rapid aggregation above 80 °C as indicated by light scattering intensity. The lectins showed simultaneous dissociation and multistate unfolding in the vicinity of GdnHCl. At pH 1.0, both the lectins exhibited molten globule like structures, which were characterized further and were found to respond in a different way towards denaturants. The results have provided valuable insights into the molecular basis of the activity and stability of the two lectins.

Biophysical principles predict fitness landscapes of drug resistance.

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype-phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.

Ultra-Rapid Glutathionylation of Ribonuclease: Is this the Real Incipit of its Oxidative Folding?

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40-50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5'-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.

Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex.

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.