FRAP and FRET Investigation of α-Synuclein Fibrillization via Liquid-Liquid Phase Separation In Vitro and in HeLa Cells.

Liquid-liquid phase separation (LLPS) acts as an important biological phenomenon in membraneless organelle formation. These phase-separated bodies can also act as nucleation centers for disease-associated amyloid formation. Fluorescence recovery after photobleaching (FRAP) is a crucial technique to analyze the material property (liquid or solid) of protein LLPS. On the other hand, Förster resonance energy transfer (FRET) is used to understand the domain-specific involvement (intermolecular interactions) of protein molecules inside the phase-separated droplets. In this protocol, we delineate mechanisms of liquid-to-solid transition of α-synuclein LLPS by using in vitro and in cell FRAP as well as in vitro FRET techniques.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Host-guest interaction aided Zinc carry and delivery by ESIPT active 2-(2'-hydroxyphenyl)benzoxazole.

The effect of solvents and supramolecular hosts on the binding of metal ion with an excited state intramolecular proton transfer (ESIPT) active fluorophore 2-(2'-hydroxyphenyl)benzoxazole (HPBO) are investigated to scrutinize a possible metal ion carry and delivery system. The fluorophore forms strong fluorescent complex with Zn2+ ion. In aqueous medium, ß-cyclodextrin (ß-CD) breaks the HPBO-Zn2+ complex and encapsulate the freed fluorophore. Hence, the initially blocked ESIPT process is restored by forming an inclusion complex with the host molecules. However, in dimethyl sulphoxide (DMSO), ß-CD does not break the complex. But cucurbit[7]uril (CB-7) breaks the complex in both DMSO and water. The tuned emission characteristics are considered for constructing different molecular logic gates. BUFFER, NOT, PASS, IMPLICATION and INHIBIT logic operations are substantiated based on Zn2+, CB-7 and ß-CD response.

Modifying the proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole by water, confinement and confined water.

The effect of water, confinement and confined water on the proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA) was investigated. Water alters the proton transfer process. At higher pH, an anion is formed in water and it undergoes intermolecular proton transfer and forms a keto tautomer. Confinement of molecule in ß-cyclodextrin affects the intramolecular proton transfer. It also prevents the intermolecular proton transfer of the anionic form. In reverse micelle, the molecule resides in the interfacial region and interacts with bound water. The intermolecular hydrogen bond of the surfactants opens the intramolecular hydrogen bond in the weaker ß-ring of bis-HPTA. It led to single tautomer emission from bis-HPTA. An increase in water amount enhances the relative amount of trans-enol, but predominantly tautomer emission is observed.

Nanoparticle and surfactant controlled switching between proton transfer and charge transfer reaction coordinates.

The reaction coordinates of a molecular photo-switch 2-(4'-diethylamino-2'-hydroxyphenyl)-1H-imidazo-[4,5-b]pyridine (DHP) was tuned with a nanoparticle and surfactant. DHP undergoes excited state intramolecular proton transfer (ESIPT) and emits normal and tautomer emissions in N,N-dimethylformamide. Silver nanoparticles suppress the ESIPT and induce twisted intramolecular charge transfer (TICT). Further addition of surfactants alters the process. Interestingly, different surfactants cause different effects. Accordingly, the luminescence characteristics are altered. The anionic surfactant sodium dodecyl sulfate (SDS) restores the ESIPT process by completely detaching the molecule from the nanoparticle. The nonionic surfactant Triton X-100 (TX-100), at lower concentration, enhances the TICT emission and the ESIPT process is also observed due to the release of some fluorophore from the nanoparticle complex. But at higher concentration the fluorophores are released completely and the ESIPT process is restored. The cationic surfactant cetyltrimethyl ammonium bromide (CTAB), at lower concentration, simply restores the ESIPT process by releasing the fluorophore. But at higher CTAB concentration, DHP enters the metalparticle-CTAB aggregate and shows enhanced ESIPT.

A detailed insight on fabricated porous chitosan in eliminating synthetic anionic dyes from single and multi-adsorptive systems with related studies.

Chitosan was fabricated via gelation method using CaBr2.xH2O/methanol solution and was studied as a potential adsorbent (MCh) in adsorbing anionic synthetic dyes like Bromophenol blue (BB), Direct blue 6 (DB) and Congo red (CR) from single (one dye species at a time) and multi (having two dyes; binary and all three dyes; tertiary) adsorptive systems. Physico-chemical modifications of MCh surface prior and post modification and dye adsorption were evaluated using scanning electron microscopy, Energy-dispersive X-ray spectroscopy, powder X-ray diffraction analysis, surface area analysis and Fourier-transformed infrared spectroscopy. Influential parameters influencing the adsorption process viz. initial pH of dye solution, MCh dosage, adsorption temperature and initial concentration of dye species were optimised. Adsorptive studies involving single adsorptive setups verified formation of sorbate's (dye species) monolayer over the sorbent's (MCh) surface via chemisorption; as established by Langmuir isotherm and pseudo-second order kinetics model analysis. Theoretical maximum adsorption capacities of MCh for BB, DB and CR was found to be 81.301 mg/g, 163.934 mg/g and 75.758 mg/g, respectively. Meanwhile, for all multi-adsorptive systems, competitive Langmuir isotherm model verified antagonistic behaviour of an individual dye over other dye adsorption over MCh surface in their respective adsorptive systems. Thermodynamics of the sorbate-sorbent interaction was exothermic, spontaneous, with elevated degree of disorderedness; concluding the interaction as thermodynamically favourable. Co-existing metal cations and anionic salts had minimal effect on MCh's adsorption efficiency. Phytotoxicity assay via germination of Vigna mungo seeds verified the efficacy of the adsorbent in eliminating the dye species from single and multi-adsorptive systems.

Light-Driven Switching between Intramolecular Proton-Transfer and Charge-Transfer States.

A molecular photoswitch, 2-(4'-diethylamino-2'-hydroxyphenyl)-1H-imidazo-[4,5-b]pyridine (DHP), with mutually independent paths of excited-state intramolecular proton transfer (ESIPT) and twisted intramolecular charge transfer (TICT) was developed. Control over these processes was attained by switching the solvents. Depending on the solvent's hydrogen-bond capacity and polarity, either one of the photoprocesses (ESIPT or TICT) or both can be triggered. Accordingly, normal and tautomer emissions, normal and TICT emissions, or triple emission of normal, tautomer, and TICT were obtained from the molecule. The emissions were resolved by fluorescence lifetime. The conclusions were established by synthesizing and studying the methoxy derivative of the molecule.

Racial disparities in COVID-19 hospitalizations do not lead to disparities in outcomes.

OBJECTIVES: The objective of the study is the identification of racial differences in characteristics and comorbidities in patients hospitalized for COVID-19 and the impact on outcomes. STUDY DESIGN: The study design is a retrospective observational study. METHODS: Data for all patients admitted to seven community hospitals in Michigan, United States, with polymerase chain reaction confirmed diagnosis of COVID-19 from March 10 to April 15, 2020 were analyzed. The primary outcomes of racial disparity in inpatient mortality and intubation were analyzed using descriptive statistics and multivariate regression models. RESULTS: The study included 336 Black and 408 White patients. Black patients were younger (62.9 ± 15.0 years vs 71.8 ± 16.4, P < .001), had a higher mean body mass index (32.4 ± 8.6 kg/m2 vs 28.8 ± 7.5, P < .001), had higher prevalence of diabetes (136/336 vs 130/408, P = .02), and presented later (6.6 ± 5.3 days after symptom onset vs. 5.4 ± 5.4, P = .006) compared with White patients. Younger Black patients had a higher prevalence of obesity (age <65 years, 69.9%) than older Black patients (age >65 years, 39.2%) and younger White patients (age < 65, 55.1%). Intubation did not reach statistical significance for racial difference (Black patients 61/335 vs. 54/406, P = .08). Mortality was not higher in Black patients (65/335 vs. 142/406 in White patients, odds ratio 0.61, 95% confidence interval: 0.37 to 0.99, 2-sided P = .05) in multivariate analysis, accounting for other risk factors associated with mortality. CONCLUSIONS: Higher prevalence of obesity and diabetes in young Black populations may be the critical factor driving disproportionate COVID-19 hospitalizations in Black populations. Hospitalized Black patients do not have worse outcomes compared with White patients.

α-Synuclein aggregation nucleates through liquid-liquid phase separation.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

Role of Cholesterol and Its Immediate Biosynthetic Precursors in Membrane Dynamics and Heterogeneity: Implications for Health and Disease.

Cholesterol is an indispensible component of cellular membranes in higher eukaryotes and plays a vital role in many cellular functions. 7-Dehydrocholesterol (7-DHC) and desmosterol represent two immediate biosynthetic precursors of cholesterol in the Kandutsch-Russell and Bloch pathways of cholesterol biosynthesis, respectively. Although 7-DHC and desmosterol differ from cholesterol merely by a double bond, accumulation of these two immediate biosynthetic precursors due to defective cholesterol biosynthesis leads to severe developmental and neurological disorders. In this context, we explored the role of cholesterol and its immediate biosynthetic precursors (7-DHC and desmosterol) on the dynamics and heterogeneity of fluid phase POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and gel phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membranes, using fluorescence lifetime distribution analysis of Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) using the maximum entropy method (MEM). We show here that the membrane interfacial dynamic heterogeneity, manifested as the width of the fluorescence lifetime distribution of Nile Red, exhibited by 7-DHC and desmosterol vastly differ from that displayed by cholesterol, particularly in fluid phase membranes. We conclude that a subtle alteration in sterol structure could considerably alter dynamic membrane heterogeneity, which could have implications in pathogenicity associated with defective cholesterol biosynthesis.

The origin of the longer wavelength emission in 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole and its analogue 2-phenylamino-5-(2-hydroxybenzono)-1,3,4-thiadiazole† ‡.

In aqueous solution, 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) was found to emit dual emission and the longer wavelength emission was assigned to the combination of aggregation and conformational change. In a number of molecules that possess an intramolecular hydrogen bond between the proton donor and the acceptor, the longer wavelength emission is often observed due to the emission from the tautomer formed by excited state intramolecular proton transfer (ESIPT). Therefore, an analogue of FABT, 2-phenylamino-5-(2-hydroxybenzono)-1,3,4-thiadiazole (PHBT), was synthesized to determine the origin of the longer wavelength emission. The luminescence of PHBT and its methoxy derivatives was studied and compared with that of FABT. Theoretical calculations were also performed on both FABT and PHBT. Based on the experimental and theoretical investigations, the nonexistence of the keto tautomer in the ground state and the origin of the longer wavelength emission are divulged.

Use of 6-Methylisoxanthopterin, a Fluorescent Guanine Analog, to Probe Fob1-Mediated Dynamics at the Stalling Fork Barrier DNA Sequences.

Fluorescent nucleic acid base mimics serve as excellent site-specific and real-time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6-methylisoxanthopterin (6-MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site-specific incorporation of this probe, we show that 6-MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double-stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double-stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6-MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double-stranded versus HJs. This work highlights the unique advantages of 6-MI as a probe when incorporated in nucleic acid frameworks.

α-Synuclein Spontaneously Adopts Stable and Reversible α-Helical Structure in Water-Less Environment.

A highly stable, spontaneous, and reversible α-helical-structure formation in recombinant and chemically modified α-synuclein protein is demonstrated for the first time in a water-less (1.5 % w/w H2 O) polymer surfactant environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that whilst native α-synuclein in aqueous solution shows a predominant unordered conformation (≈64 %), mixing with polyethylene glycol based anionic polymer surfactant (PS) and removing water reveals a 25 % unordered, 25 % α-helical, and 27 % ß-sheet structure. Interestingly, bioconjugation of native α-synuclein with a diamine molecule, to increase the positive charge on the protein chain, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) water-less liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25-30 w/w% of water in the system. Moreover, the α-helix shows an extraordinary temporal stability (>6 months) in a waterless environment.

Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein.

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (IMG), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy IMG state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the IMG molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the "wrong turn" to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.

Tweaking the proton transfer triggered proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole.

The proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA), a fluorophore proficient in self-aided twin proton transfer, has been explored in the presence of dimethylformamide (DMF). The proton transfer is completely altered in the presence of DMF (compared to that in other solvents). Bis-HPTA forms a hydrogen bonded complex with DMF molecules, which shift the conformer equilibrium. Theoretical calculations predicted that the DMF complexes of bis-HPTA-II and bis-HPTA-III are more stable than the complexes of other conformers. Upon excitation, bis-HPTA-II transfers a proton to a DMF molecule to form an anion and bis-HPTA-III transfers a proton to a ring nitrogen to form a keto form. Silver particles reverse the effect of DMF. They regenerate bis-HPTA-I by removing the intermolecular hydrogen bond and thereby they reestablish the proton transfer triggered proton transfer (PTTPT).

Proton transfer triggered proton transfer: a self-assisted twin excited state intramolecular proton transfer.

The double excited state intramolecular proton transfer (ESIPT) of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA), a molecule possessing two intramolecular hydrogen bonded donor-acceptor pairs, has been investigated. The molecule undergoes not only a single ESIPT, but also a rare twin ESIPT. The most interesting fact is that initially, only one acid-base pair is ESIPT active and the other pair is ESIPT inactive. The first proton transfer triggers the proton transfer in the second acid-base pair by creating appropriate conditions. This new type of sequential proton transfer is labeled as 'proton transfer triggered proton transfer' (PTTPT). The PTTPT has been demonstrated by steady state and time resolved fluorescence techniques. It was further substantiated by theoretical data and using a chemically modified system 3-(2-hydroxyphenyl)-5-(2-methoxyphenyl)-1H-1,2,4-triazole.

Bioactive compound from Aspergillus terreusDMTMGK004 synergistically contributes towards potential anti-pathogenicity.

This study focused on the evaluation of fungal compound for their anti-pathogenic potential against respiratory pathogens. Soil samples were collected from various geographical regions in Madurai, fungal strain was isolated and identified as Aspergillus terreusDMTMGK004 (MGK004). Secondary metabolites were extracted and evaluated for antioxidant potential. It exhibited significantly high anti-proliferative property against gastric adenocarcinoma (AGS) cell lines. Antimicrobial activity against Gram positive (Streptococcus pneumoniae) and Gram negative (Klebsiella pneumoniae and Haemophilus influenzae) respiratory pathogens were analysed and the minimum inhibitory concentration (MIC) values were determined. Furthermore, the time-killing assay illustrated that the metabolite eliminates 50% of the vegetative cells within few hours of the treatment. From the spectral data, the major functional groups present in the compound were determined as carbonyl group and phenolic hydroxyl group which contribute towards its bioactivity. The compound significantly depreciates the production of extracellular polysaccharides which results in the weakening of biofilm architecture and resistance towards serum killing and phagocytosis. It also induced cell membrane damage which leads to protein and nucleic acid leakage. Hence, the results of the present study could provide a better insinuation towards the formulation of new drug targeting respiratory pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The ubiquitous fungi Aspergillus terreus is well known for its secondary metabolite production. The fungus was evaluated for production of antagonistic molecule to reduce the growth of infectious agents causing respiratory infections. It exhibited the biological means of antioxidant, anti-proliferative and anti-pathogenic compound production. The compound exhibits killing effect against respiratory pathogens within two hours. It induced cell membrane damage leading to protein and nucleic acid leakage. It significantly reduced the production of extracellular polysaccharides. The results provide needed information to design innovative strategies for targeting pathogenic factors of the respiratory pathogens instead of killing it precisely.

Fluorescence spectroscopy for revealing mechanisms in biology: Strengths and pitfalls.

This article describes the basic principles of steady-state and time-resolved fluorescence. The formal equivalence of the two methodologies is described first, followed by the extra advantages of time-resolved methods in revealing population heterogeneity in complex systems encountered in biology. Several examples from the author's work are described in support of the above contention. Finally, several misinterpretations and pitfalls in the interpretation of fluorescence data and their remedy are described.

Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the ß-sheet.

Perturbation of cationic equilibrium by cucurbit-7-uril.

The effect of cucurbit-7-uril (CB-7) on a cationic mixture with same charge has been investigated by studying monocationic mixtures of 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine (DMAPIP-b) and 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-c]pyridine (DMAPIP-c). The pKa of both the guests increases in CB-7. DMAPIP-b forms all three monocations in the ground and the excited states in aqueous as well as in CB-7 solution. However, CB-7 shifts the equilibrium more towards the less polar MC2 and MC3. On the other hand, DMAPIP-c exists only as MC1 and MC3 in aqueous solution, however, in CB-7 it exists not only as MC1 and MC3 but also as MC2 in CB-7 in the ground state. In the excited state, as in aqueous solution MC1 forms MC2 by biprotonic phototautomerism in CB-7. The association constants of monocations suggest a pyridyl nitrogen position dependence. All the MC-CB-7 complexes are optimized by density functional theory (DFT).