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Diabetes represents a major health problem, involving a severe imbalance of blood sugar levels, which can disturb the nerves, eyes, kidneys, and other organs. Diabes management involves several synthetic drugs focused on improving insulin sensitivity, increasing insulin production, and decreasing blood glucose levels, but with unclear molecular mechanisms and severe side effects. Natural chemicals extracted from several plants such as Gymnema sylvestre, Momordica charantia or Ophiopogon planiscapus Niger have aroused great interest for their anti-diabetes activity, but also their hypolipidemic and anti-obesity activity. Here, we focused on the anti-diabetic activity of a few natural and synthetic compounds, in correlation with their pharmacokinetic/pharmacodynamic profiles, especially with their blood-brain barrier (BBB) permeability. We reviewed studies that used bioinformatics methods such as predicted BBB, molecular docking, molecular dynamics and quantitative structure-activity relationship (QSAR) to elucidate the proper action mechanisms of antidiabetic compounds. Currently, it is evident that BBB damage plays a significant role in diabetes disorders, but the molecular mechanisms are not clear. Here, we presented the efficacy of natural (gymnemic acids, quercetin, resveratrol) and synthetic (TAK-242, propofol, or APX3330) compounds in reducing diabetes symptoms and improving BBB dysfunctions. Bioinformatics tools can be helpful in the quest for chemical compounds with effective anti-diabetic activity that can enhance the druggability of molecular targets and provide a deeper understanding of diabetes mechanisms.

Background:: Synthetic compounds with pharmaceutical applications in brain disorders are daily designed and synthesized, with well first effects but also seldom severe side effects. This imposes the search for alternative therapies based on the pharmaceutical potentials of natural compounds. The natural compounds isolated from various plants and arthropods venom are well known for their antimicrobial (antibacterial, antiviral) and antiinflammatory activities, but more studies are needed for a better understanding of their structural and pharmacological features with new therapeutic applications. Objectives:: Here we present some structural and pharmaceutical features of natural compounds isolated from plants and arthropods venom relevant for their efficiency and potency in brain disorders. We present the polytherapeutic effects of natural compounds belonging to terpenes (limonene), monoterpenoids (1,8-cineole) and stilbenes (resveratrol), as well as natural peptides (apamin, mastoparan and melittin). Methods:: Various experimental and in silico methods are presented with special attention on bioinformatics (natural compounds database, artificial neural network) and cheminformatics (QSAR, drug design, computational mutagenesis, molecular docking). Results:: In the present paper we reviewed: (i) recent studies regarding the pharmacological potential of natural compounds in the brain; (ii) the most useful databases containing molecular and functional features of natural compounds; and (iii) the most important molecular descriptors of natural compounds in comparison with a few synthetic compounds. Conclusion:: Our paper indicates that natural compounds are a real alternative for nervous system therapy and represents a helpful tool for the future papers focused on the study of the natural compounds.

Proteins undergo continuous changes under the action of various intrinsic and extrinsic factors, leading to alteration of several intracellular metabolic pathways and the development of various clinical disorders. Non-enzymatic glycosylation is one of the main factors responsible for the progression of diabetic complications and the aging process. Although there are currently many effective therapies in the prevention and treatment of these diseases, in the last decade, there has been an increasing trend of replacing synthetic drugs by natural compounds, in order to reduce the side effects that may occur and the production costs. It is well known that aspirin (acetylsalicylic acid) inhibits the glycation process of serum proteins by acetylating N-terminal amino groups and lysine residues in their structure. Therefore, the main purpose of our research was to analyze the non-enzymatic glycation and fructation process of hemoglobin through spectrometric and electrophoretic techniques, in order to reveal how this process could influence the three-dimensional structure and biological function of the protein, and the effect of some natural aspirin-like compounds on the peroxidase and esterase activity of hemoglobin during fructose and glucose binding. In this way, a preliminary phytochemical characterization of a bark extract of white willow (Salix alba) was performed in order to evaluate the content of total phenolics, flavonoids, and salicylic derivatives, as well as the antioxidant activity. Then, human erythrocytes isolated from whole peripheral blood were incubated with different concentrations of fructose/glucose (10, 50, 100 mM) and S. alba extract for 5, 7, 10 and 14 days. The results obtained from the THz spectra confirmed that fructose was more reactive than glucose, so the glycation process took place more slowly than fructation. Also, the presence of S. alba extract showed an antiglycosylating effect, but not a total inhibition of the glycation process. In addition, enzymatic determinations proved that willow bark extract restored the peroxidase and esterase activities to the control levels. Our data indicated that salicylic compounds can be successfully used as substitutes for aspirin, one of the main synthetic compounds with anti-inflammatory and anti-glycosylating roles. Salicin, salicylic acid and other salicylic compounds possess strong antioxidant properties, which give them the ability to participate in the glycosylation process to block the formation of advanced glycation end products (AGEs).

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe damage to the respiratory system. With no specific treatment to date, it is crucial to identify potent inhibitors of SARS-CoV-2 Chymotrypsin-like protease (3CLpro) that could also modulate the enzymes involved in the respiratory damage that accompanies SARS-CoV-2 infection. Here, flavones isolated from Scutellaria baicalensis (baicalein, baicalin, wogonin, norwogonin, and oroxylin A) were studied as possible compounds in the treatment of SARS-CoV-2 and SARS-CoV-2-induced acute lung injuries. Methods: We used structural bioinformatics and cheminformatics to (i) identify the critical molecular features of flavones for their binding activity at human and SARS-CoV-2 enzymes, (ii) predict their drug-likeness and leader-likeness features, (iii) calculate their pharmacokinetic profile, with an emphasis on toxicology, (iv) predict their pharmacodynamic profiles, with the identification of their human body targets involved in the respiratory system injuries, and (v) dock the ligands to SARS-CoV-2 3CLpro. Results: All flavones presented appropriate drug-like and kinetics features, except for baicalin. Flavones could bind to SARS-CoV-2 3CLpro at a similar site, but interact slightly differently with the protease. Flavones&rsquo, pharmacodynamic profiles predict that (i) wogonin strongly binds at the cyclooxygenase2 and nitric oxide synthase, (ii) baicalein and norwogonin could modulate lysine-specific demethylase 4D-like and arachidonate 15-lipoxygenase, and (iii) baicalein, wogonin, norwogonin, and oroxylin A bind to SARS-CoV-2 3CLpro. Conclusions: Our results propose these flavones as possible potent drugs against respiratory damage that occurs during SARS-CoV-2 infections, with a strong recommendation for baicalein.

Background: During the current SARS-CoV-2 pandemic, the identification of effective antiviral drugs is crucial. Unfortunately, no specific treatment or vaccine is available to date. Objective: Here, we aimed to predict the interactions with SARS-CoV-2 proteins and protein targets from the human body for some flavone molecules (kaempferol, morin, pectolinarin, myricitrin, and herbacetin) in comparison to synthetic compounds (hydroxychloroquine, remdesivir, ribavirin, ritonavir, AMD-070, favipiravir). Methods: Using MOE software and advanced bioinformatics and cheminformatics portals, we conducted an extensive analysis based on various structural and functional features of compounds, such as their amphiphilic field, flexibility, and steric features. The structural similarity analysis of natural and synthetic compounds was performed using Tanimoto coefficients. The interactions of some compounds with SARS-CoV-2 3CLprotease or RNA-dependent RNA polymerase were described using 2D protein-ligand interaction diagrams based on known crystal structures. The potential targets of considered compounds were identified using the SwissTargetPrediction web tool. Results: Our results showed that remdesivir, pectolinarin, and ritonavir present a strong structural similarity which may be correlated to their similar biological activity. As common molecular targets of compounds in the human body, ritonavir, kaempferol, morin, and herbacetin can activate multidrug resistance-associated proteins, while remdesivir, ribavirin, and pectolinarin appear as ligands for adenosine receptors. Conclusion: Our evaluation recommends remdesivir, pectolinarin, and ritonavir as promising anti- SARS-CoV-2 agents.

The current COVID-19 pandemic initiated an unprecedented response from clinicians and the scientific community in all relevant biomedical fields. It created an incredible multidimensional data-rich framework in which deep learning proved instrumental to make sense of the data and build models used in prediction-validation workflows that in a matter of months have already produced results in assessing the spread of the outbreak, its taxonomy, population susceptibility, diagnostics or drug discovery and repurposing. More is expected to come in the near future by using such advanced machine learning techniques to combat this pandemic. This review aims to unravel just a small fraction of the large global endeavors by focusing on the research performed on the main COVID-19 targets, on the computational weaponry used in identifying drugs to combat the disease, and on some of the most important directions found to contain COVID-19 or alleviating its symptoms in the absence of specific medication.

TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure.

Two different types of nanoparticles (silicon dioxide and titanium dioxide) were selected within this study in order to analyze the interaction with bovine and human serum albumin. These particles were characterized by transmission and scanning electron microscopy (TEM and SEM), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDXS). In addition, the hydrodynamic size and the zeta potential were measured for all these nanoparticles. The serum proteins were incubated with the nanoparticles for up to one hour, and the albumin adsorption on the particle surface was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The effect induced on the secondary structure of proteins was analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that albumin adsorbed on the surface of both types of nanoparticles, but in different quantities. In addition, we noticed different changes in the structure of albumin depending on the physicochemical properties of each type of particle tested. In conclusion, our study provides a comparative analysis between the different characteristics of nanoparticles and the protein corona formed on the particle surface and effects induced on protein structure in order to direct the development of &ldquo, safe-by-design&rdquo, nanoparticles, as their demands for research and applications continue to increase.

Antimicrobial peptides, also called body defense peptides, are chemical structures widely distributed across the animal and vegetal kingdoms that have a fundamental role as part of the immune system. These peptides are used against a wide range of pathogens, such as Gram-negative and - positive bacteria, fungi and viruses, etc. Their action spectrum makes them important for the pharmaceutical industry, as they could represent templates for the design of new and more potent structures by using drug design and drug delivery systems. Here we present the antimicrobial activity against Bacillus subtilis (expressed as minimal inhibitory concentration values) for 33 mastoparan analogs and their new derivatives by quantitative structure-activity relationship method (2D, aligned and also non-aligned 3D-QSAR). We establish the contribution to antimicrobial activity of molecular descriptors like hydrophobicity, hydrogen bond donor and steric hindrance, correlated with contributions from the membrane environment (sodium, potassium, chloride ions). Also the studies of HIV-1 fusion inhibitor sifuvirtide and its analogs are presented in context of interaction with lipid structures during fusion and delivery of these drugs.

Due to their outstanding properties, quantum dots (QDs) received a growing interest in the biomedical field, but it is of major importance to investigate and to understand their interaction with the biomolecules. We examined the stability of silicon QDs and the time evolution of QDs – protein corona formation in various biological media (bovine serum albumin, cell culture medium without or supplemented with 10% fetal bovine serum-FBS). Changes in the secondary structure of BSA were also investigated over time. Hydrodynamic size and zeta potential measurements showed an evolution in time indicating the nanoparticle-protein interaction. The protein corona formation was also dependent on time, albumin adsorption reaching the peak level after 1 hour. The silicon QDs adsorbed an important amount of FBS proteins from the first 5 minutes of incubation that was maintained for the next 8 hours, and diminished afterwards. Under protein-free conditions the QDs induced cell membrane damage in a time-dependent manner, however the presence of serum proteins attenuated their hemolytic activity and maintained the integrity of phosphatidylcholine layer. This study provides useful insights regarding the dynamics of BSA adsorption and interaction of silicon QDs with proteins and lipids, in order to understand the role of QDs biocorona.

The nonenzymatic attachment of sugars to proteins, namely glycation, is accelerated under diabetic conditions. Monitoring the glycated human serum albumin (HSA) levels gives the short term variation of glucose concentration in diabetic patients blood. Therefore, a significant effort was made to measure glycated HSA, including by spectroscopic methods such as Raman. Here we used THz spectroscopy to monitor HSA glycation in time (over 5, 7 and 11 weeks). Different sugar types have different reactivity; therefore we also addressed the reducing sugar influence on glycation by performing in vitro HSA glycation by both glucose and fructose. Since residues protonation state influences their susceptibility for glycation, we incubated HSA with sugars at two pH values: 7 and 8. Our results show that THz absorption decreases with the incubation time of HSA with sugars. At the incubation times we considered, the most significant differences were obtained on HSA samples glycated using glucose. Differences between samples glycated by glucose and by fructose show that glycation by glucose is a slower process. At pH 7, glycation by glucose is slower than at pH 8, while glycation by fructose is slightly faster at pH 7 than at pH 8. Glycated HSA models with different degrees of glycation were built by molecular modeling. Simulated THz spectra of the models are in good agreement with the experimental data. All these show that THz spectroscopy could monitor the progression of glycation in time and that it is sensitive to reducing sugars or pH value used in the glycation process.

Terahertz (THz) spectroscopy is a promising technique for the study of protein structure and internal flexibility. Here, we used THz spectroscopy and molecular modeling for bovine serum albumin (BSA) structure investigation. BSA molecule was built using homology modeling methods and 30 different more relaxed models were obtained by molecular dynamics simulations of the hydrated protein. As the experimental and simulated THz spectra are linear, we compared them by comparing the slopes of the lines that fit them. Six BSA structures had slope values in the range given by the slope of the experimental spectrum $$\pm $$ 0.2 and a total of sixteen BSA structures had slope values in the 0.6 interval near the experimental slope value. BSA average structures were calculated over the six and the sixteen identified BSA molecules. Based on the similarity with the crystal structure of BSA, we validated the average structure calculated over the sixteen BSA conformations. The comparison with the crystal structure showed that the structure validated using THz spectroscopy is a coarse model of BSA, as its root-mean-square deviation relative to the crystal structure is 1.9 A. The regions from our model that present the highest deviation from the crystal structure are exterior loops. The results presented here show that using THz spectroscopy and molecular modeling is a promising approach to determine the structure of proteins.

Nuclear radiation can seriously damage biological samples. Some radiation effects are the production of reactive radicals resulted from water radiolysis and random bond breakings, protein fragmentation, cross-linking or aggregation resulted from random ionizations in proteins. Similar effects were described in the case of X-rays irradiation. Therefore, we assumed that not only nuclear radiation, but also the lower energy X-rays produce flexibility changes in proteins that can be addressed by Terahertz (THz) spectroscopy. Here we simulated and compared the THz spectra of two Torpedo californica acetylcholinesterase structures that were exposed to synchrotron radiation (hard X-ray of ~13 keV) over different periods of time. Our results show a clear difference between simulated absorptions, which allows us to conclude that radiation induced changes in proteins structures and flexibility should be visible on THz spectra. By detecting even subtle effects produced by radiation softer than nuclear radiation, THz spectroscopy should be useful in detecting effects of higher energy radiation that is part of the chemical, biological, radiological and nuclear (CBRN) threat.

Up-converting nanoparticles are dielectric crystalline particles doped with rare-earth ions such as Yb3+, Er3+, Tm3+, Ho3+, Nd3+, etc. When excited in infrared, they emit visible radiation. Used as markers, they present significant advantages in comparison to traditional fluorophores: sharp emission lines, superior photostability, resistence to photobleaching, no blinking and lack of toxicity. Infrared radiation is less harmful to cells avoiding tissue degradation, minimizes auto-fluorescence from endogenous biocomponents offering a good signal-to-background ratio and penetrates tissues deeply. In spite of the great advantages of using up-converting nanoparticles for biomedical applications, there are still some limitations. These refer to identification of optimal size suited for specific samples, prevention of aggregation, water stability/dispersibility, optical efficiency and biocompatibility. This chapter reviews principal characteristics of up-converting nanoparticles and issues related to their use in biomedical applications.

Background Alzheimer's disease (AD) therapy is based on several natural and synthetic compounds that act as acetylcholinesterase (AChE) and N-methyl-D-aspartate receptor (NMDA) ligands that have limited efficiency in relieving AD symptoms. Recent studies show that inhibitors isolated from Mentha spicata L. subsp. spicata are promising for AD therapy. Objective We aimed to identify novel and more potent phytopharmaceutical compounds for AD treatment by taking into account the compounds from Mentha spicata L. subsp. spicata essential oil. Method We generated structure-activity relationship (SAR) models that predict the biological activities of 14 Mentha spicata L. subsp. spicata compounds on AChE and NMDA by comparing their molecular features with those of the three conventional ligands: donepezil, galantamine and memantine. Results The most relevant descriptors for predicting the biological activities of considered compounds are solvent accessible area and their subdivided, hydrophobicity, energy of frontier molecular orbitals and counts of the aromatic ring and rotatable bounds. 1,8-cineole, the main compound from Mentha spicata L. subsp. spicata essential oil, resulted to be similar with memantine and dissimilar with donepezil in respect to hidrophobicity (logP1,8-cineole=2.95, logPmemantine=2.81, logPdonepezil=4.11), the energy of LUMO (eLUMO1,8-cineole=3.01 eV, eLUMOmemantine=3.35 eV, eLUMOdonepezil=-0.35 eV) and the solvent accessible surface areas over all hydrophobic (SA_H1,8-cineole= 350 A2, SA_Hmemantine= 358 A2, SA_Hdonepezil= 655 A2) or polar atoms (SA_P1,8-cineole= 4 A2, SA_Pmemantine=10 A2, SA_Pdonepezil=44.62 A2). Conclusion Our results point towards 1,8-cineole as a good candidate for NMDA antagonism, with a weaker AChE inhibitory effect. Our results may be useful in establishing new therapeutic strategies for neurological disorders.

The standardization of HIV-1 strains for vaccine tests include the use of viral reference panels. We determined a series of pharmacological descriptors (molecular surfaces, volumes, electrostatic energies, solvation energies, number of atoms, number of hydrogen donors or acceptors and number of rigid bonds) for the gp120 CD4-binding sites structures in the unliganded state from a reference panel of 60 diverse strains of HIV-1. We identified the descriptors that varied significantly between the strains, the outliner strains for each descriptor set and the possible correlations between the descriptors. Our results improve the knowledge about gp120, its molecular and possible neutralization properties.

The function of the epithelial Na+ channel from the apical membrane of many Na+ transporting epithelia is modulated by various chemical compounds from the extracellular space, such as heavy metals, protons or chloride ions. We have studied the effect of extracellular Cd2+ on the function of the epithelial Na+ channel (ENaC) in heterologously expressed Xenopus laevis oocytes and Na+-transporting epithelia. We assayed channel function as the amiloride-sensitive sodium current (I(Na)). Cd2+ rapidly and voltage-independently inhibited INa in oocytes expressing αβγ Xenopus ENaC (xENaC). The extracellular Cd2+ inhibited Na+ transport and showed no influence on ENaC trafficking, as revealed by concomitant measurements of the transepithelial current, conductance and capacitance in Na+-transporting epithelia. Instead, amiloride inhibition was noticeably diminished in the presence of Cd2+ on the apical membrane. Using molecular modeling approaches, we describe the amiloride binding sites in rat and xENaC structures, and we present four putative binding sites for Cd2+. These results indicate that ENaC functions as a sensor for external Cd2+.

Pentaerythritol tetranitrate (PETN) reductase is an enzyme produced by bacteria (Enterobacter cloacae PB2) that have the ability to use nitroaromatic (trinitrotoluene – TNT, picric acid) or nitroester (glycerol trinitrate – GTN, PETN) explosives as a sole nitrogen source for growth. The reaction of PETN reductase comprises a reductive and an oxidative half-reaction, explosives being degraded during the oxidative half-reaction. Since the side chains of the residues that form the active site of PETN reductase assume different conformations during the two half-reactions, we investigated whether THz spectroscopy could discriminate between the different states of PETN reductase and whether their discrimination could be useful in the detection of explosives and consequent monitoring of explosives degradation process. In order to answer these questions, we simulated and compared the THz spectra of the hydrated enzyme in the reduced state, in the oxidized state bound to an inert substrate, namely a thiocyanate ion and in the oxidized state bound to picric acid. Our results show that PETN reductase structures in reduced and oxidized states present a significantly different THz absorption, the enzyme in reduced state being the one that absorbs more THz radiation. In oxidized state, the enzyme bound to thiocyanate absorbs less THz radiation then the enzyme bound to picric acid. By discriminating between the conformations of PETN reductase during the two half-reactions involved in explosives degradation, THz spectroscopy should allow the evaluation of explosives degradation state.

In normal cells, the accuracy of chromosome segregation which assures cells euploidy depends on mitosis mechanics and on proper functioning of a specific complex of proteins represented by the error-checking spindle assembly checkpoint (SAC). SAC proteins are deeply involved in correct cell divisions, but some of these, such as mitotic arrest-deficient proteins (Mad1 and Mad2), are critical. Mad1 and Mad2 are involved in preventing “wrong” cellular divisions which lead to cellular aneuploidy and are recognized as inductors of genetic disorders, as well as activators of oncoproteins. To clarify aneuploidy involvement in the evolution of cancer or other genetic disorders, structural and functional specificity of spindle checkpoint proteins have been analyzed, but the process is still poorly understood. In order to better understand SAC proteins involvement in initiation of cancer and other genetic disorders, here we review studies that conducted to relevant structural and functional information regarding these proteins. The results of these studies suggest that minor changes in structure and functionality of SAC proteins are able to generate aneuploidy. Therefore, a deeper understanding of Mad1 and Mad2 structural changes obtained by experimental and theoretical studies could open new perspectives of genetic medicine.

Within medicinal chemistry nowadays, the so-called pharmaco-dynamics seeks for qualitative (for understanding) and quantitative (for predicting) mechanisms/models by which given chemical structure or series of congeners actively act on biological sites either by focused interaction/therapy or by diffuse/hazardous influence. To this aim, the present review exposes three of the fertile directions in approaching the biological activity by chemical structural causes: the special computing trace of the algebraic structure-activity relationship (SPECTRAL-SAR) offering the full analytical counterpart for multi-variate computational regression, the minimal topological difference (MTD) as the revived precursor for comparative molecular field analyses (CoMFA) and comparative molecular similarity indices analysis (CoMSIA); all of these methods and algorithms were presented, discussed and exemplified on relevant chemical medicinal systems as proton pump inhibitors belonging to the 4-indolyl,2-guanidinothiazole class of derivatives blocking the acid secretion from parietal cells in the stomach, the 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine congeners’ (HEPT ligands) antiviral activity against Human Immunodeficiency Virus of first type (HIV-1) and new pharmacophores in treating severe genetic disorders (like depression and psychosis), respectively, all involving 3D pharmacophore interactions.

Terahertz Time-Domain Spectroscopy (THz-TDS) is a new technique in studying the conformational state of molecules. Cell membranes are important structures in the interaction with extra cellular entities. Their principal building blocks are lipids, amphiphilic molecules that spontaneously self-assemble when in contact with water. In this work we report the use of THz-TDS in transmission mode to examine the behavior of supported phospholipid bilayers (SPBs) within the frequency range of 0.2 THz to 3 THz. SPBs were obtained by vesicle adsorption method involving the spread of a suspension (50-100 μl) of small unilamellar vesicles (SUVs) or multilamellar vesicles (MLVs) dissolved in PBS (phosphate buffer solution) on a support of silicon wafers. Both SUVs and MLVs were obtained from dipalmitoyl phosphatidylcholine (DPPC) and lecithin by using the thin-film hydration method. Broadband THz pulses are generated and detected using photoconductive antennas optically excited by a femtosecond laser pulse emitted from a self-mode locked fiber laser at a wavelength of 780 nm with a pulse widths of 150 fs. THz-TDS was proven to be a useful method in studying SPBs and their hydration states. The absorption coefficient and refractive index of the samples were calculated from THz measurements data. The THz absorption spectra for different lipids in SPBs indicate specific absorption frequency lines. A difference in the magnitude of the refractive index was also observed due to the different structure of supported lipid bilayers. The THz spectrum of DPPC was obtained by using theoretical simulations and then the experimental and theoretical THz spectra were compared.

Bovine serum albumin (BSA) is the most abundant protein in bovine plasma; its three dimensional structure is yet unknown. We investigated the structure and dynamics of BSA in lyophilized samples, in 10% w/w and 50% w/w BSA aqueous solutions using THz spectroscopy and molecular modeling. THz spectra were recorded with a spectral resolution of 7.4 GHz. Theoretical spectra were simulated using a structural model of BSA based on the homology with the known structure of human serum albumin (HSA). The agreement between simulated THz spectra and THz spectra recorded experimentally allowed us to validate the BSA model and the solution models. Based on these models we investigated the flexibility of dry BSA and of BSA with one hydration layer. The hydrated structure of BSA is less flexible than the structure free of water molecules, except for residues 54 - 104 that are more mobile in the hydrated structure. We also investigated the fluctuations of the water molecules within the first hydration layer and identified two groups of water molecules: one that exhibits small fluctuations and one of highly mobile water molecules. These molecules are associated to highly mobile regions from the proteins and move in positive correlation with the neighboring protein regions. We also propose a BSA dimerization model in which the molecules strongly interact. The fluctuations of the BSA monomers and of their first hydration layer were investigated. The two molecules display similar fluctuation patterns, but one of them is slightly more flexible.

The low frequency collective movements of proteins associated with biologically relevant conformational transitions are situated in the terahertz (THz) spectral region. THz spectroscopy experiments have revealed that each molecular species has a unique absorption pattern in THz domain. We used time-domain THz spectroscopy (TDS) to study the conformation and flexibility of bovine serum albumin (BSA), a protein whose 3D structure is unknown. We performed THz spectroscopy experiments on lyophilized BSA. Theoretical spectra were obtained by normal modes analysis performed on BSA structures generated by homology modeling and molecular dynamics simulations. The agreement between experimental and theoretical data allowed us to validate the model of BSA and also to gain insight into BSA vibrations in THz domain.

THz spectroscopy was used to investigate bovine serum albumin (BSA) flexibility in conditions ranging from lyophilised BSA to dilute 10% w/w BSA solution and highly concentrated 50% w/w BSA solutions. The simulated THz spectra of dry and hydrated BSA are in good agreement with the experiments.

Macromolecular crowding is a common intracellular phenomenon that causes conformational changes of proteins and protein association. We investigated these macromolecular crowding effects on a highly concentrated BSA solution using THz spectroscopy and molecular modeling. We modeled several BSA 50% w/w solutions comprising two BSA molecules in a water box and selected a single model based on the agreement with THz experiments. We further modeled BSA association at concentrations higher than 50% w/w and selected a possible dimer model based on the strength of the interaction between the two proteins. The flexibility of the BSA dimer was compared with the flexibility of BSA from the solution. Monomeric BSA from the solution model presents mobile regions scattered through all the structure, with differences of disposition and extent between the two molecules. Dimerization changes BSA flexibility, as the two molecules from the dimer present compact regions of both high flexibility and low flexibility. The low flexibility regions include their interaction sites.

THz spectroscopy is a very powerful tool in investigating the vibrational modes and conformational transitions of biomolecules. Here we have used THz spectroscopy to study the vibrational spectrum of bovine serum albumin, a protein whose 3D structure is yet unknown. Experimental data were fitted with theoretical spectra obtained by normal modes analysis of BSA structures generated by homology modeling.

The native cellular environment represents a crowded system comprising high concentrations of soluble molecules that interact mostly in a nonspecific manner. Some of the macromolecular crowding effects occurring in biological media are conformational changes and macromolecular associations. Most of our knowledge on protein folding and protein-protein interactions was acquired from experiments on proteins in dilute solutions or from theoretical models of isolated proteins in either explicit or implicit solvent. Here we present a 50% w/w bovine serum albumin (BSA) solution model that comprises two solute molecules included in a single water box. We determined the vibration spectrum of the 50% w/w BSA solution using THz spectroscopy and we calculated the theoretical THz spectrum. We observed a good correlation between the experimental and theoretical spectra for the frequency range of 0.3 - 1.5 THz. We also investigated the contribution of each BSA molecule to the solution THz spectrum by simulating THz spectra of the two BSA molecules from the solution model and water, each accounting for a 50% w/w BSA solution. The spectra appear to be similar. As the two molecules in our solution model have different conformations, we investigated the importance of the apparently insignificant differences between simulated THz spectra of the two proteins. We found that the differences should be considered significant, as they reflect differences between the flexibility of the two BSA molecules.

Diaryldiamidines, a class of DNA minor groove binders used clinically since the 1940s in the treatment of tropical parasitoses such as trypanosomiasis, leishmaniasis, pneumocystis pneumonia and babesiosis, have been recently found to exert specific blocking effects on acid-sensing ion channels (ASIC), raising hopes to develop new small molecule neuroprotective agents for ischemic stroke, which involves acidosis-mediated activation of ASIC1a and heteromeric ASIC1a/2b channels. Therefore we have thoroughly investigated the modes of interaction of diminazene with ASIC1a via whole-cell and single-channel recordings on outside-out patches excised from HEK293 cells of ASIC1a transient currents evoked by brief acidic pulses (pH 6.0). Data idealization and model-dependent fitting were performed with custom hidden Markov model maximum likelihood algorithms. We proved that the 3 subconductance levels are due to partial block by calcium of the permeation pathway, and expanded our previous 8-state linear connectivity gating model (Marin et al. 2008 Channels 2(6):1-10) including 3 subconductance states for each of the 2 gating modes. Single-channel recordings with diminazene 0.6 or 3 μM indicated a mechanism of open channel block. By exponential fits of the distributions of blocked and unblocked periods for each gating mode we computed binding and unbinding rates and dissociation constants of 0.13 and 0.27 μM for gating modes 2 and 1, respectively. Using atomic resolution models of human ASIC1a in the open conformations corresponding to the 2 gating modes, we found via molecular docking several intra- and intersubunit binding sites for diminazene. One of these sites, assumed to be responsible for the state-dependent block, features protrusion of the drug in the transmembrane domain permeation pathway. As expected, for this blocking site the interaction energy is higher in the symmetrical open conformation of the second gating mode than in the asymmetrical open conformation of the first gating mode.