Selective vitamins as potential options for dietary therapeutic interventions: In silico and In vitro insights from mutant C terminal fragment of FGA.

We have used an integrated computational approach to explore the role of vitamin C and vitamin D in preventing aggregation of Fibrinogen A alpha-chain (FGActer) protein responsible for renal amyloidosis. We modelled structures of E524K / E526K mutants of FGActer protein and examined the potential interactions of these mutants with vitamin C and vitamin D3. Interaction of these vitamins at the amyloidogenic site may prevent the intermolecular interaction required for amyloid formation. The binding free energy values of vitamin C and vitamin D3 for E524K FGActer and E526K FGActer are - 67.12 ± 30.46 kJ/mole and - 79.45 ± 26.12 kJ/mol, respectively. Experimental studies using Congo red absorption, aggregation index studies and AFM imaging show encouraging results. The AFM images of E526K FGActer contained more extensive and higher protofibril aggregates, whereas, in the presence of vitamin D3, small monomeric and oligomeric aggregates were observed. Overall, the works provide interesting results about vitamin C and D role in preventing renal amyloidosis.

Vitamin C and Vitamin D3 show strong binding with the amyloidogenic region of G555F mutant of Fibrinogen A alpha-chain associated with renal amyloidosis: proposed possible therapeutic intervention.

G555F mutant of Fibrinogen A alpha-chain (FGA) is reported to be associated with kidney amyloidosis. In the current study, we have modelled the G555F mutant and examined the mutation's effect on the structural and functional level. We have also docked Vitamin C and D3 on the mutant's amyloidogenic region to identify if these vitamins can bind amyloidogenic regions. Further, we analyzed if they could prevent or modulate amyloid formation by stopping critical interactions in amyloidogenic regions in FGA. We used the wild type FGA model protein as a control. Our docking and molecular dynamics simulation results indicate stronger Vitamin D3 binding than Vitamin C to the amyloidogenic region of the mutant protein. The RMSD, radius of gyration, and RMSF values were higher for the G555F mutant than the FGA wild type protein. Overall, the results support these vitamins' potential as a therapeutic and anti-amyloidogenic agent for FGA renal amyloidosis.

Drugs repurposing against SARS-CoV2 and the new variant B.1.1.7 (alpha strain) targeting the spike protein: molecular docking and simulation studies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) is responsible for the global COVID-19 pandemic and millions of deaths worldwide. In December 2020, a new alpha strain of SARS-CoV2 was identified in the United Kingdom. It was referred to as VUI 202012/01 (Alpha strain modelled under investigation, 2020, month 12, number 01). The interaction between spike protein and ACE2 receptor is a prerequisite for entering virion into the host cell. The present study is focussed on the spike protein of the SARS-COV 2, involving the comparison of binding affinity of new alpha strain modelled spike with previous strain spike (PDB ID:7DDN) using in silico molecular docking, dynamics and simulation studies. The molecular docking studies of the alpha strain modelled spike protein confirmed its higher affinity for the ACE2 receptor than the spike protein of the dominant strain. Similar computational approaches have also been used to investigate the potency of FDA approved drugs from the ZINC Database against the spike protein of new alpha strain modelled and old ones. The drug molecules which showed strong affinity for both the spike proteins are then subjected to ADME analysis. The overall binding energy of Conivaptan (-107.503 kJ/mol) and Trosec (-94.029 kJ/mol) is indicative of their strong binding affinities, well supported by interactions with critical residues.

Molecular and clinical insights into protein misfolding and associated amyloidosis.

The misfolding of normally soluble proteins causes their aggregation and deposition in the tissues which disrupts the normal structure and function of the corresponding organs. The proteins with high ß-sheet contents are more prone to form amyloids as they exhibit high propensity of self-aggregation. The self aggregated misfolded proteins act as template for further aggregation that leads to formation of protofilaments and eventually amyloid fibrils. More than 30 different types of proteins are known to be associated with amyloidosis related diseases. Several aspects of the amyloidogenic behavior of proteins remain elusive. The exact reason that causes misfolding of the protein and its association into amyloid fibrils is not known. These misfolded intermediates surpass the over engaged quality control system of the cell which clears the misfolded intermediates. This promotes the self-aggregation, accumulation and deposition of these misfolded species in the form of amyloids in the different parts of the body. The amyloid deposition can be localized as in Alzheimer disease or systemic as reported in most of the amyloidosis. The amyloidosis can be of acquired type or familial. The current review aims at bringing together recent updates and comprehensive information about protein amyloidosis and associated diseases at one place.

Biophysical characterization of fibroblast growth factor homologous factor-1b (FHF-1b): sodium dodecyl sulfate promotes two state folding.

The current article describes the biophysical characterization and folding studies of fibroblast growth factor homologous factor-1b (FHF-1b) in comparison with acidic fibroblast growth factor (FGF-1). Our data indicates that FHF-1 is significantly more stable than FGF-1. The folding mechanism of these two proteins seems to be different although they share high degree of sequence and structural similarity. FHF-1 unfolds through stable intermediate state while unfolding of FGF-1 is two-state. Interestingly, low concentration of sodium dodecyl sulfate (SDS) drives the folding pathway of FHF-1b to two-state.

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding.

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states.

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.

Crystallization and preliminary X-ray analysis of cryptolepain, a novel glycosylated serine protease from Cryptolepis buchanani.

Cryptolepain is a stable glycosylated novel serine protease purified from the latex of the medicinally important plant Cryptolepis buchanani. The molecular weight of the enzyme is 50.5 kDa, as determined by mass spectrometry. The sequence of the first 15 N-terminal resides of the protease showed little homology with those of other plant serine proteases, suggesting it to be structurally unique. Thus, it is of interest to solve the structure of the enzyme in order to better understand its structure-function relationship. X-ray diffraction data were collected from a crystal of cryptolepain and processed to 2.25 A with acceptable statistics. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.78, b = 108.15, c = 119.86 A. The Matthews coefficient was 2.62 A(3) Da(-1) with one molecule in the asymmetric unit. The solvent content was found to be 53%. Structure determination of the enzyme is under way.

A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization.

A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.

Snapshots of protein folding problem: implications of folding and misfolding studies.

Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii.

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.