Electrostatic Modulation of Intramolecular and Intermolecular Interactions during the Formation of an Amyloid-like Assembly.

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro-in silico approach to ascertain the structure-dynamics-stability-aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM), under a bivariate solution condition in terms of pH and salt concentration. Under acidic pH conditions, the native TDP-43tRRM protein creates an aggregation-prone entropically favorable partially unfolded conformational landscape due to enthalpic destabilization caused by the protonation of the buried ionizable residues and consequent overwhelming fluctuations of selective segments of the sequence leading to anti-correlated movements of the two domains of the protein. The evolved fluffy ensemble with a comparatively exposed backbone then easily interacts with incoming protein molecules in the presence of salt via typical amyloid-aggregate-like intermolecular backbone hydrogen bonds with a considerable contribution originating from the dispersion forces. Subsequent exposure to excess salt at low pH conditions expedites the aggregation process via an electrostatic screening mechanism where salt shows preferential binding to the positively charged side chain. The applied target observable-specific approach complementarity unveils the hidden information landscape of an otherwise complex process with unquestionable conviction.

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43.

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.

Shapeshifter TDP-43: Molecular mechanism of structural polymorphism, aggregation, phase separation and their modulators.

TDP-43 is a nucleic acid-binding protein that performs physiologically essential functions and is known to undergo phase separation and aggregation during stress. Initial observations have shown that TDP-43 forms heterogeneous assemblies, including monomer, dimer, oligomers, aggregates, phase-separated assemblies, etc. However, the significance of each assembly of TDP-43 concerning its function, phase separation, and aggregation is poorly known. Furthermore, how different assemblies of TDP-43 are related to each other is unclear. In this review, we focus on the various assemblies of TDP-43 and discuss the plausible origin of the structural heterogeneity of TDP-43. TDP-43 is involved in multiple physiological processes like phase separation, aggregation, prion-like seeding, and performing physiological functions. However, the molecular mechanism behind the physiological process performed by TDP-43 is not well understood. The current review discusses the plausible molecular mechanism of phase separation, aggregation, and prion-like propagation of TDP-43.

Multistep molecular mechanism of amyloid-like aggregation of nucleic acid-binding domain of TDP-43.

TDP-43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid-like assembly of the functional nucleic acid-binding domain of TDP-43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveal that aggregation occurs in four distinct steps-very fast, fast, slow, and very slow. The "very fast" change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the "fast step" to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the "slow" step, which, consequently, assembles in the "very slow" step to form an amyloid-like assembly. The spectroscopic properties of the amyloid-like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid-like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics.

Dry Molten Globule-Like Intermediates in Protein Folding, Function, and Disease.

The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding-unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.

Effect of In Vitro Solvation Conditions on Inter- and Intramolecular Assembly of Full-Length TDP-43.

Cellular stress is a major cause of neurodegenerative diseases. In particular, in amyotrophic lateral sclerosis (ALS), around 90% of the cases are believed to occur due to aggregation and misfolding of TDP-43 protein in neurons due to aging and chronic environmental stress. However, the physicochemical basis of how TDP-43 senses the change in solvation conditions during stress and misfolds remains very poorly understood. We show here that the full-length human TDP-43 can exist in equilibrium with multiple structural states. The equilibrium between these states is highly sensitive to changes in solvation conditions. We show that upon thermal and pH stress, amyloidogenic oligomers can form amyloid-like fibrils. However, the internal structure of the fibril depends upon the physicochemical nature of stress. Our results present a physical basis of the effect of solvation conditions on inter- and intramolecular assembly formation of TDP-43 and reconcile why the nature and the internal structure of the aggregated form have been found to be different when extracted from the brain of different ALS patients.

The native state conformational heterogeneity in the energy landscape of protein folding.

The native structure of proteins is central to various functions performed by cells. A vital part of the structure-function paradigm of proteins is their inherent flexibility and dynamics. The dynamic interconversion between the conformational substates in the heterogeneous native state basin of the energy landscape enables a single protein molecule to perform multiple functions. The dynamics among the substates are assisted by the motion of different structural elements of a protein out of which side-chains of amino acids hold a significant position due to their involvement in various functions such as molecular recognition and dynamic allostery. This review briefly describes the origin of conformational heterogeneity in the native state ensemble and the motions of different structural modules that assist the equilibrium dynamics of the conformational substates. The review then centers the discussion on conformational heterogeneity due to side-chain movements in proteins, the experimental methods to detect and characterize them and their role in performing multiple functions.

Kinetic modeling and statistical optimization of submerged production of anti-Parkinson's prodrug L-DOPA by Pseudomonas fluorescens.

L-DOPA, a precursor of dopamine, is the drug of choice for Parkinson's disease, which persists due to decreased levels of dopamine in the brain. Present study emphasis the microbial production of L-DOPA rather than the biotransformation of L-DOPA by L-tyrosine. The production of L-DOPA by bacterial isolates had gained more acceptance due to its more straightforward extraction and downstream processes. Pseudomonas fluorescens was used to produce the L-DOPA in a bioreactor system under submerged condition. The design of experiment-based Taguchi orthogonal array method was adopted for the optimization of production. L-9 orthogonal array using the analysis of mean approach was used to study the effect of different factors viz NaCl, lactose, tryptone, and inducer on the microbial production of L-DOPA. The method mentioned above is less time consuming and does not require any harsh chemicals, proving it to be an eco-friendly process. After optimizing selected factors, i.e., NaCl (1.2 g/l), lactose (1.5 g/l), tryptone (4 g/l), and inducer (0.1 g/l), 16.9 % of enhancement in L-DOPA production with 66.6% of process cost saving was observed. The production of L-DOPA was increased from 3.426 ± 0.08 g/l to 4.123 ± 0.05 g/l after optimization. Subsequently, unstructured kinetic models were adopted to simulate the fermentation kinetics and understand the metabolic process. Fisher' F test and determination coefficients (R2) confirmed that the Velhurst-Pearl logistic equation, Luedeking-Piret equation, and modified Luedeking-Piret equation was best fitted with the biomass production, product formation, and substrate utilization, respectively.

Assessment of antioxidant and cytotoxicity activities against A-549 lung cancer cell line by synthesized reduced graphene oxide nanoparticles mediated by Camellia sinensis.

Camellia sinensis (green tea leaves) which acts as a reducing agent was used for the reduction of graphene oxide (GO) to obtain reduced graphene oxide (RGO). Anionic surfactant SDS was used to enhance the stability of synthesized reduced graphene oxide nanoparticles. Characterized reduced graphene oxide nanoparticle grain size was calculated to be 3.92 nm from the X-ray diffraction method, whereas zeta potential was measured - 35.23 ± 5.45 mV at room temperature. Antioxidant and cell cytotoxicity against A-549 lung carcinoma cells were also studied. Phytochemical content of Camellia sinensis imparts feasible DPPH activity of 85.98 ± 2.49% against RGO, whereas ABTS scavenging activity was found to be 88.87 ± 1.74% followed by measurement of the total phenolic content of 842 ± 13.33 µg/gm. RGO at concentration 400 µg/ml showed an optimum level of hemolysis at pH 7.4 (4.92 ± 1.20%) than pH 5.6 (11.15 ± 0.03%). Cytotoxicity activity studied by MTT assay of RGO on A-549 lung carcinomas cells was compared with drug doxorubicin. The bandgap energy of RGO was calculated to be 3.97 eV from absorption data, hence reveals the generation of oxidative stress in the A-549 lung cancer cell line. Thus, the surfactant and phytochemicals found in Camellia sinensis enhanced the stability of RGO, thereby providing enough energy to destabilize the target cells without affecting healthy cells, hence suggests its role in therapeutics application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03015-z.

Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.

Nutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43tRRM) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, ß form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-ß transition. We observed that D105A and H256Q resembled TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the ß form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.

A pH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue.

The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pH-dependent change in the stability of HSA was observed in the near native pH range (pH 6.0-9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.

Production and purification of bioflocculants from newly isolated bacterial species: a comparative decolourization study of cationic and anionic textile dyes.

Bioflocculant-producing bacteria were isolated from various water reservoirs and sediments of the water treatment plant. Four promising strains were identified by standard biochemical methods and 16s rRNA gene sequencing. Bioflocculants were produced in a batch bioreactor of 3 L under optimized conditions. Fourier transformed infrared spectroscopy and scanning electron microscopy (SEM) were used to confirm the chemical and morphological nature of bioflocculants. Anionic and cationic textile dyes congo red (CR) and rhodamine-B (RB) decolourization efficiency by ethanol precipitated bioflocculants were accessed under different values of pH, temperature, dose of flocculant and presence of monovalent, divalent and trivalent cations. Bioflocculants of all the four isolates were found to be highly efficient in decolourization of dye from an aqueous medium with the removal rate up to 99.56%. The removal rate of CR and RB from aqueous medium was largely influenced by the physiochemical condition of the solution viz. pH, temperature, concentration of ions and dose of flocculants. The microbial bioflocculants are biodegradable and highly stable as well as possess abroad range of pH, temperature and ions tolerance range. So, they may be economical and can be greener substitutes for the present harsh chemical-based wastewater effluent treatment methods.

Preparation, characterization and application of curcumin based polymeric bio-composite for efficient removal of endotoxins and bacterial cells from therapeutic preparations.

Polymer science offers a great insight and a new research dimension for biomedical applications. The synthesis of polymeric materials by the physical ways provides several advantages over the conventional chemical methods. It is though expansive but less toxic, stable, and efficiently reproducible. In the present report, electrospinning was used for bio-composite preparation. The bio-composite was developed using polyvinyl alcohol (PVA) and curcumin. The electrospun fiber bio-composite were analyzed for antibacterial activity, bacterial filtration capability, and endotoxin elimination. The bio-composite was analyzed for physical structure and properties using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray analysis (EDX), and Fourier Transform Infra-Red spectroscopy (FT-IR). PVA solely was not able to exhibit any of the antibacterial or endotoxin removal properties. However, the curcumin-based bio-composite was found to be bactericidal and endotoxin eliminator. The bio-composite was able to remove 100% of endotoxin and nearly 100% of the bacterial cells. The endotoxin removal properties of bio-composite were found to be excellent fit under Langmuir curve with a R2 value of 0.98. Additionally, the effect of bio-composite was also studied over protein content in the sample and L-asparaginase activity. However, the effect observed was negligible.

Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process.

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K2HPO4 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µm 0.104 h-1, Xm 6g/L P0 1.7U/mL Pm 8.2 U/mL YX/S 4 g-cell/g-glucose µPm 0.35 h-1 qp 5.46 U/g/h YP/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a ß-sheet rich "ß form" in at least two distinct steps. The ß form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ ß conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

Production, purification and kinetic characterization of glutaminase free anti-leukemic L-asparaginase with low endotoxin level from novel soil isolate.

Anti-leukemic enzyme L-asparaginase despite having significant applicability in medicine, holds side effects attributed to glutaminase activity and endotoxin content. Glutaminase activity proves to be toxic to non-tumor cells as glutamine is an essential amino acid. Endotoxin illicit the production of vasoactive amines and induce septic shock. Hence there is a need for glutaminase free L-asparaginase with minimum endotoxin level. The report aims at the development of a downstream process for purification of glutaminase free L-asparaginase and subsequent endotoxin removal. Producing bacteria were isolated from various soil samples and screened initially for asparaginase and glutaminase activity. The glutaminase free L-asparaginase producing bacteria were identified as Bacillus altitudinis. Production of L-asparaginase was optimized. The optimum medium comprised of comprising Lactose (1.5 g/L), NaCl (1.2 g/L), Yeast extract (5 g/L), L-asparagine (20 g/L) with pH 7.0 and incubation time of 18 h. Kinetic parameters Km and Vmax were computed to be 9.09x10-2M and 0.09 M/S. L-asparaginase Purification was achieved with a specific activity of 800 U/mg of enzyme. Molecular weight of the purified L-asparaginase was determined to be around 35 KDa using SDS-PAGE. The developed process also brought down the endotoxin content below the FDA recommended level. The endotoxin content of the purified enzyme was determined to be 0.015EU/mL.

Coexistence of Gonadal Dysgenesis and Mullerian Agenesis in a Female with 46 XX Karyotype: A Case Report.

Gonadal dysgenesis is a rare genetically heterogeneous disorder characterized by underdeveloped ovaries with consequent, impuberism, primary amenorrhea, and hypergonadotropic hypogonadism. Mullerian agenesis or Mayer­Rokitansky­Kuster­Hauser syndrome is characterized by congenital aplasia of the uterus and the upper part (2/3) of the vagina in a woman with normal development of secondary sexual characteristics and a normal 46 XX karyotype. The association of gonadal dysgenesis and Mayer-Rokitansky-Kuster-Hauser syndrome is very rare and appears to be coincidental. We report a case of a 24-years old woman who presented with primary amenorrhea. The endocrine study revealed hypergonadotrophic hypogonadism. The karyotype was normal, 46,XX. Internal genitalia could not be identified on the pelvic ultrasound and pelvic MRI. There were no other morphological malformations. Keywords: Gonadal dysgenesis; Mayer Rokitansky Kuster Hauser syndrome; Mullerian agenesis; primary amenorrhea; 46,XX.

Correction: A dry molten globule-like intermediate during the base-induced unfolding of a multidomain protein.

Correction for 'A dry molten globule-like intermediate during the base-induced unfolding of a multidomain protein' by Nirbhik Acharya et al., Phys. Chem. Chem. Phys., 2017, 19, 30207-30216.

Slow Motion Protein Dance Visualized Using Red-Edge Excitation Shift of a Buried Fluorophore.

It has been extremely challenging to detect protein structures with a dynamic core, such as dry molten globules, that remain in equilibrium with the tightly packed native (N) state and that are important for a myriad of entropy-driven protein functions. Here, we detect the higher entropy conformations of a human serum protein, using red-edge excitation shift experiments. We covalently introduced a fluorophore inside the protein core and observed that in a subset of native population, the side chains of the polar and buried residues have different spatial arrangements than the mean population and that they solvate the fluorophore on a timescale much slower than the nanosecond timescale of fluorescence. Our results provide direct evidence for the dense fluidity of protein core and show that alternate side-chain packing arrangements exist in the core that might be important for multiple binding functions of this protein.

The Folding and Aggregation Energy Landscapes of Tethered RRM Domains of Human TDP-43 Are Coupled via a Metastable Molten Globule-like Oligomer.

Stress-induced misfolding and intraneuronal aggregation of the highly conserved nucleic acid binding protein TDP-43 (transactive response DNA binding protein 43 kDa) and its fragments have been implicated in amyotrophic lateral sclerosis and several other neurodegenerative diseases. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. TDP-43 is a four-domain protein and performs the essential nucleic acid binding function with the help of its two tandem RNA recognition motif domains naturally tethered by a linker (called here the tethered RRM domain of TDP-43 or TDP-43tRRM). Here, we show that the monomeric native form of TDP-43tRRM remains in a pH-dependent and reversible thermodynamic equilibrium with a protonated, nanosized, 40-meric form (the A form). Under the stress-like low-pH condition, the A form becomes predominantly populated. In the A form, protein molecules have restricted dynamics of surface side-chain residues but native-like secondary structure. This self-assembled form possesses a loosely packed core in which the intrinsically disordered and aggregation-prone regions are in the proximity. The A form is metastable and swiftly aggregates into a highly stable amyloid-like protofibrillar form (ß form) mediated by the disorder-to-order transition of intrinsically disordered regions upon small environmental perturbations. Interestingly, the A form and the ß form are not formed when TDP-43tRRM is bound to DNA, indicating that the nucleic acid binding regions of the protein participate in their formation. Our results reveal how the energy landscapes of folding and aggregation of TDP-43tRRM are coupled by a metastable molten-globule like oligomeric form and modulated by stress-like conditions.