TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments.

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.

Phosphorylation of Troponin I finely controls the positioning of Troponin for the optimal regulation of cardiac muscle contraction.

Troponin is the Ca2+ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca2+ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH2-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH2-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH2-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca2+ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH2-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH2-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca2+ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH2-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.

Characterization of the L29Q Hypertrophic Cardiomyopathy Mutation in Cardiac Troponin C by Paramagnetic Relaxation Enhancement Nuclear Magnetic Resonance.

The key events in regulating muscle contraction involve the troponin (Tn) heterotrimeric protein complex in which the binding to and release of Ca2+ from the highly conserved troponin C (TnC) subunit trigger a series of structural changes within Tn, and the other thin filament proteins, to result in contraction. In the heart, the control of contraction and relaxation events can be altered by many single-point mutations that may result in cardiomyopathy and sometimes sudden cardiac death. Here we have examined the structural effects of one hypertrophic cardiomyopathy mutation, L29Q, on Ca2+-induced structural transitions within whole TnC. This mutation is of particular interest as several physiological and structural studies have indicated that the response of TnC to Ca2+ binding is altered in the presence of the L29Q mutation, but the structural nature of these changes continues to be debated. In addition, little is known about the effect of this mutation in the Ca2+ free state. Here we have used paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) to assess the structural effects arising from the L29Q mutation. PRE-NMR distances obtained from a nitroxide spin-label at Cys84 showed that the L29Q mutation perturbs the structure of the TnC N-domain in the presence and absence of Ca2+, with a more "open" TnC N-domain observed in the apo form. In addition, binding of Ca2+ to the TnC-L29Q construct triggers a change in the orientation between the two domains of TnC. Together, these structural perturbations, revealed by PRE-NMR, provide insight into the pathogenesis of this mutation.

Development of remdesivir repositioning as a nucleotide analog against COVID-19 RNA dependent RNA polymerase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative representative of a severe respiratory illness resulted in widespread human infections and deaths in nearly all of the countries since late 2019. There is no therapeutic FDA-approved drug against SARS-CoV-2 infection, although a combination of anti-viral drugs is directly being practiced in some countries. A broad-spectrum of antiviral agents are being currently evaluated in clinical trials, and in this review, we specifically focus on the application of Remdesivir (RVD) as a potential anti-viral compound against Middle East respiratory syndrome (MERS) -CoV, SARS-CoV and SARS-CoV-2. First, we overview the general information about SARS-CoV-2, followed by application of RDV as a nucleotide analogue which can potentially inhibits RNA-dependent RNA polymerase of COVs. Afterwards, we discussed the kinetics of SARS- or MERS-CoV proliferation in animal models which is significantly different compared to that in humans. Finally, some ongoing challenges and future perspective on the application of RDV either alone or in combination with other anti-viral agents against CoVs infection were surveyed to determine the efficiency of RDV in preclinical trials. As a result, this paper provides crucial evidence of the potency of RDV to prevent SARS-CoV-2 infections.Communicated by Ramaswamy H. Sarma.

The expression level of angiotensin-converting enzyme 2 determines the severity of COVID-19: lung and heart tissue as targets.

Researchers have reported some useful information about the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to CoV disease 2019 (COVID-19). Several studies have been performed in order to develop antiviral drugs, from which a few have been prescribed to patients. Also, several diagnostic tests have been designed to accelerate the process of identifying and treating COVID-19. It has been well-documented that the surface of host cells is covered by some receptors, known as angiotensin-converting enzyme 2 (ACE2), which mediates the binding and entry of CoV. After entering, the viral RNA interrupts the cell proliferation system to activate self-proliferation. However, having all the information about the outbreakof the SARS-COV-2, it is not still clear which factors determine the severity of lung and heart function impairment induced by COVID-19. A major step in exploring SARS-COV-2 pathogenesis is to determine the distribution of ACE2 in different tissues . In this review, the structure and origin of CoV, the role of ACE2 as a receptor of SARS-COV-2 on the surface of host cells, and the ACE2 distribution in different tissues with a focus on lung and cardiovascular system have been discussed. It was also revealed that acute and chronic cardiovascular diseases (CVDs) may result in the clinical severity of COVID-19. In conclusion, this review may provide useful information in developing some promising strategies to end up with a worldwide COVID-19 pandemic.Communicated by Ramaswamy H. Sarma.

Constructing a structural model of troponin using site-directed spin labeling: EPR and PRE-NMR.

The relative ease of introducing a paramagnetic species onto a protein, and advances in electron paramagnetic resonance (EPR) over the past two decades, have established spin labeling as a vital structural biology technique for revealing the functional workings of the troponin muscle regulatory complex-an ~80 kDa heterotrimeric protein switch for turning on striated muscle contraction. Through the site-directed spin labeling (SDSL) of cysteine residues at key sites in troponin, a molecular-level understanding of the troponin muscle regulatory system across all levels of structural hierarchy has been achieved. Through the application of EPR, mobility and accessibility trends in the EPR signals of the spin labels attached to consecutive residues can reveal the secondary structure of troponin elements and also help map the interaction between subunits. Distance restraints calculated from the interspin interactions between spin label pairs have helped with building a structural model of the troponin complex. Further, when SDSL is paired with NMR, paramagnetic relaxation enhancement (PRE)-NMR has been used to obtain high-resolution structural detail for both intra- and interdomain interactions in troponin and revealed details of protein conformational changes and dynamics accompanying troponin function. In this review, we provide an overview of the SDSL labeling methodology and its application towards building a dynamic structural model of the multi-subunit troponin complex which details the calcium-induced conformational changes intimately linked to muscle regulation. We also describe how the SDSL method, in conjunction with EPR or NMR, can be used to obtain insights into structural perturbations to troponin caused by disease-causing mutations.

Paclitaxel inhibited lysozyme fibrillation by increasing colloidal stability through formation of "off-pathway" oligomers.

Protein fibrillation is a challenging issue in medicine, causing many diseases, and an impediment to pharmaceutics and protein industry. Many chemicals, especially polyphenol compounds and aromatic small molecules, have been widely used as an effective strategy to combat protein fibril formation. Hence, understanding mechanisms of fibrillation inhibition and contributing forces in this process are significant. In this study, the inhibitory effect of paclitaxel on lysozyme fibrillation was investigated with respect to thermal and colloidal stability. Fibrillation was monitored with ThT fluorescence, circular dichroism, and AFM; paclitaxel-lysozyme interaction with isothermal titration calorimetry and docking; thermal and colloidal stability with differential scanning calorimetry and zeta-pulse, respectively. Paclitaxel inhibited lysozyme fibrillation, and interacted with lysozyme through hydrogen bonds and van der Waals' interactions. The viability of PC12 cells retrieved as a result of fibrillation inhibition by paclitaxel. Hydrophobic forces dominantly shielded the aggregation-prone region of lysozyme and suppressed the effective interactions between lysozyme monomers. Although paclitaxel did not affect lysozyme's thermal stability, it increased lysozyme's colloidal stability by either increasing the surface charge density or charge distribution on lysozyme. In conclusion, our results suggest a model for paclitaxel's inhibitory role through two complementary steps driving to "off-pathway" oligomer formation and attenuation of fibril formation.

Sodium dodecyl sulphate modulates the fibrillation of human serum albumin in a dose-dependent manner and impacts the PC12 cells retraction.

Protein aggregation is impacted by many factors including temperature, pH, and the presence of surfactants, electrolytes, and metal ions. The addition of sodium dodecyl sulphate (SDS) at different concentrations may play a significant role in the human serum albumin (HSA) fibrillation pathway. Here the heat induction of HSA fibrillation incubated with different concentrations of SDS was evaluated using a variety of techniques. These included ThT fluorescence, Congo red absorbance, circular dichroism, dynamic light scattering, and atomic force microscopy (AFM). To explore HSA surface properties, the surface tension of solutions was measured using Du Noüy Ring method tensiometry. In addition, the criteria of neurite outgrowth and complexity were monitored by exposing PC12 cells to different forms of HSA amyloid intermediates. ThT fluorescence kinetic studies indicated that SDS at low concentrations induced more fibrillation of HSA, while SDS at high concentrations inhibited the fibrillation of HSA. At higher SDS concentrations hydrophobic forces had a significant role whereas at lower SDS concentrations electrostatic forces were dominant. The cell culture studies demonstrated the significant impact of SDS concentration on HSA fibrillation and subsequent neuronal cell morphology. The HSA incubated with low concentrations of SDS inhibited neurite outgrowth and complexity of the PC12 cells, whereas high concentrations of SDS had lesser effect. Thus, SDS acts as a salt at lower concentrations, while at higher concentrations acts as a chaperon, with significant impact on fibrillation of HSA.

Inhibition study on insulin fibrillation and cytotoxicity by paclitaxel.

Alzheimer, a neurodegenerative disease, and a large variety of pathologic conditions are associated with a form of protein aggregation known as amyloid fibrils. Since fibrils and prefibrillar intermediates are cytotoxic, numerous attempts have been made to inhibit fibrillation process as a therapeutic strategy. Peptides, surfactants and aromatic small molecules have been used as fibrillation inhibitors. Here we studied the effects of paclitaxel, a polyphenol with a high tendency for interaction with proteins, on fibrillation of insulin as a model protein. The effects of paclitaxel on insulin fibrillation were determined by Thioflavin T fluorescence, Congo red absorbance, circular dichroism and atomic force microscopy. These studies indicated that paclitaxel considerably hindered nucleation, and therefore, fibrillation of insulin in a dose-dependant manner. The isothermal titration calorimetry studies showed that the interaction between paclitaxel and insulin was spontaneous. In addition, the van der Waal's interactions and hydrogen bonds were prominent forces contributing to this interaction. Computational results using molecular dynamic simulations and docking studies revealed that paclitaxel diminished the polarity of insulin dimer and electrostatic interactions by increasing the hydrophobicity of its dimer state. Furthermore, paclitaxel reduced disrupting effects of insulin fibrils on PC12 cell's neurite outgrowth and complexity, and enhanced their survival.

Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells.

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.

Highly efficient immobilization of beta-lactoglobulin in functionalized mesoporous nanoparticles: a simple and useful approach for enhancement of protein stability.

The immobilization of ß-lactoglobulin-B (BLG-B) onto the amine-functionalized KIT-6 [n-PrNH(2)-KIT-6], which has average pore diameter around 6.5 nm, was studied. [n-PrNH(2)-KIT-6] proved to be highly effective agent for BLG-B adsorption. UV-visible spectroscopy studies demonstrated that the immobilized BLG-B was less prone to thermally induced aggregation than the free protein. Circular dichroism (CD) spectra of free and immobilized BLG-B were recorded and significant differences in both the backbone and aromatic regions of the spectra were observed upon thermic stress. The obtained results showed that structural elements of the immobilized BLG-B are kept strongly together, making the protein more resistant to heat denaturation. The melting temperatures of the free and immobilized BLG-B were measured by far-UV CD, which showed 19 °C higher heat resistance of the immobilized BLG-B compared with its free form. Acrylamide quenching of fluorescence of free and immobilized forms of BLG-B as a function of temperature revealed that the immobilized BLG-B was more resistant to Trp quenching. Therefore immobilization of BLG-B onto [n-PrNH(2)-KIT-6] is accompanied by favorable structural stability of BLG-B in the confined space.