Protein drugs are important therapeutic agents however; they may degrade during formulation processing. The objective of this study was to investigate the correlation between secondary structure alterations and the retentions of biological activity of protein upon the application of thermal stress. Catalase, horseradish peroxidase and α- chymotrypsin were employed as model proteins. Each protein was heated in a solid and solution state at a temperature of 70 °C for 1 h. Attenuated total reflectance Fourier transform infrared spectroscopy, size-exclusion chromatography and biological activity assay were performed. Results showed that heat-exposure of protein solids at 70 °C caused minimum changes in secondary structure and biological activity was almost retained. However, thermal exposure of protein aqueous solution induced significant changes in the secondary structure indicated by area overlap values and caused considerable reduction in the biological activity. The changes in secondary structures were found to be in full alignment with the loss of biological activity for both protein solids as well as aqueous solutions. Catalase lost entire biological activity upon heating in the solution state. In conclusion, the findings of the present study indicate a direct correlation between protein secondary structure alterations and the retention of biological activity which can be taken into account during the development and delivery of protein drugs formulations.
Mitochondrial Subunit ND1 (mtND1) gene is involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). Alteration of the electron transport components by mutations in mtDNA may compromise the normal electron flow. This could lead to an increase of bifurcation and generation of superoxidase radicals and increase oxidative stress in various types of cancer cells. Genomic DNA was extracted from thirty matched primary colorectal tumour tissues and matching non-tumour tissues. Blood samples were obtained from twenty-five normal people. The mtNDI coding region was amplified by step-down PCR. The purified products were then subjected to direct sequencing and subsequently, the DNA sequences obtained were compared with the revised Cambridge Reference Sequence (rCRS) and MITOMAP. From the analysis, the mtND1 gene showed 11 (45.8%) different mutations and also 13 (54.2%) polymorphisms. The heteroplasmic mutation A4123A/G (I273I/V) might have a pathogenic significance as it fulfills various pathogenic criteria. Three mutations, T3394C (Y30H), A3434G (Y43C) and C3497T (A64V) which occur in a highly conserved region were likely to alter the structure and function of the ND1 protein. We suggest that these mutations, and in combination with the polymorphic variance in mtDNA, may cause slight changes that generate subtly higher levels of toxic reactive oxygen species (ROS).
Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.
Schistosomiasis is a major parasitic disease of humans, second only to malaria in its global impact. The disease is caused by digenean trematodes that infest the vasculature of their human hosts. These flukes are limited externally by a body wall composed of a syncytial epithelium, the apical surface membrane of which is a parasitism-adapted dual membrane complex. Annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the first structural and immunobiochemical characterization of an annexin from Schistosoma mansoni. The crystal structure of annexin B22 confirms the presence of the previously predicted α-helical segment in the II/III linker and reveals a covalently linked head-to-head dimer. From the calcium-bound crystal structure of this protein, canonical type II, type III and B site positions are occupied, and a novel binding site has been identified. The dimer arrangement observed in the crystal structure suggests the presence of two prominent features, a potential non-canonical membrane binding site and a potential binding groove opposite to the former. Results from transcriptional profiling during development show that annexin B22 expression is correlated with life stages of the parasite that possess the syncytial tegument layer, and ultrastructural localization by immuno-electron microscopy confirms the occurrence of annexins in the tegument of S. mansoni. Data from membrane binding and aggregation assays indicate the presence of differential molecular mechanisms and support the hypothesis of annexin B22 providing structural integrity in the tegument.
The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 A resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation.
Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.
DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.
The recognition of protein folds is an important step in the prediction of protein structure and function. Recently, an increasing number of researchers have sought to improve the methods for protein fold recognition. Following the construction of a dataset consisting of 27 protein fold classes by Ding and Dubchak in 2001, prediction algorithms, parameters and the construction of new datasets have improved for the prediction of protein folds. In this study, we reorganized a dataset consisting of 76-fold classes constructed by Liu et al. and used the values of the increment of diversity, average chemical shifts of secondary structure elements and secondary structure motifs as feature parameters in the recognition of multi-class protein folds. With the combined feature vector as the input parameter for the Random Forests algorithm and ensemble classification strategy, we propose a novel method to identify the 76 protein fold classes. The overall accuracy of the test dataset using an independent test was 66.69%; when the training and test sets were combined, with 5-fold cross-validation, the overall accuracy was 73.43%. This method was further used to predict the test dataset and the corresponding structural classification of the first 27-protein fold class dataset, resulting in overall accuracies of 79.66% and 93.40%, respectively. Moreover, when the training set and test sets were combined, the accuracy using 5-fold cross-validation was 81.21%. Additionally, this approach resulted in improved prediction results using the 27-protein fold class dataset constructed by Ding and Dubchak.
There are a lot of factors and conditions to be considered by tour and travel companies when designing quality product due to the fact that the product being sold is intangible and their ultimate goal is to sustain customers' loyalty. Fuzzy Logic Controller (FLC) has been observed to be compatible to this 'intangible' factor thus giving better result when compared to other methods. By using FLC, all communications are clear and have precise meaning.
The ability to predict local structural features of a protein from the primary sequence is of paramount importance for unraveling its function in absence of experimental structural information. Two main factors affect the utility of potential prediction tools: their accuracy must enable extraction of reliable structural information on the proteins of interest, and their runtime must be low to keep pace with sequencing data being generated at a constantly increasing speed. Here, we present NetSurfP-2.0, a novel tool that can predict the most important local structural features with unprecedented accuracy and runtime. NetSurfP-2.0 is sequence-based and uses an architecture composed of convolutional and long short-term memory neural networks trained on solved protein structures. Using a single integrated model, NetSurfP-2.0 predicts solvent accessibility, secondary structure, structural disorder, and backbone dihedral angles for each residue of the input sequences. We assessed the accuracy of NetSurfP-2.0 on several independent test datasets and found it to consistently produce state-of-the-art predictions for each of its output features. We observe a correlation of 80% between predictions and experimental data for solvent accessibility, and a precision of 85% on secondary structure 3-class predictions. In addition to improved accuracy, the processing time has been optimized to allow predicting more than 1000 proteins in less than 2 hours, and complete proteomes in less than 1 day.
One of the most important goals in bioinformatics is the ability to predict tertiary structure of a protein from its amino acid sequence. In this paper, new feature groups based on the physical and physicochemical properties of amino acids (size of the amino acids' side chains, predicted secondary structure based on normalized frequency of β-Strands, Turns, and Reverse Turns) are proposed to tackle this task. The proposed features are extracted using a modified feature extraction method adapted from Dubchak et al. To study the effectiveness of the proposed features and the modified feature extraction method, AdaBoost.M1, Multi Layer Perceptron (MLP), and Support Vector Machine (SVM) that have been commonly and successfully applied to the protein folding problem are employed. Our experimental results show that the new feature groups altogether with the modified feature extraction method are capable of enhancing the protein fold prediction accuracy better than the previous works found in the literature.
The stability of biocatalysts is an important criterion for a sustainable industrial operation economically. T1 lipase is a thermoalkalophilic enzyme derived from Geobacillus zalihae strain T1 (T1 lipase) that was isolated from palm oil mill effluent (POME) in Malaysia. We report here the results of high temperatures molecular dynamics (MD) simulations of T1 lipase in explicit solvent. We found that the N-terminal moiety of this enzyme was accompanied by a large flexibility and dynamics during temperature-induced unfolding simulations which preceded and followed by clear structural changes in two specific regions; the small domain (consisting of helices alpha3 and alpha5, strands beta1 and beta2, and connecting loops) and the main catalytic domain or core domain (consisting of helices alpha6- alpha9 and connecting loops which located above the active site) of the enzyme. The results suggest that the small domain of model enzyme is a critical region to the thermostability of this organism.
Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an "immature" Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.
Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.
Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.
Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, "low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also", we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06-0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure.
Conformational analysis of champedak galactose-binding (CGB) lectin under different urea concentrations was studied in phosphate-buffered saline (pH 7.2) using far-ultraviolet circular dichroism (far-UV CD), tryptophan (Trp) fluorescence and ANS fluorescence. In all cases, CGB lectin displayed a two-step, three-state transition. The first transition (from the native state to the intermediate state) started at ∼2.0 M urea and ended at ∼4.5 M urea, while the second transition (from the intermediate state to the completely denatured state) was characterized by the start- and end-points at ∼5.75 M and ∼7.5 M urea, respectively, when analyzed by the emission maximum of Trp fluorescence. A marked increase in the Trp fluorescence, ANS fluorescence and -CD values at 218 nm (-CD218 nm) represented the first transition, whereas a decrease in these parameters defined the second transition. On the other hand, emission maximum of the Trp fluorescence showed a continuous increase throughout the urea concentration range. Transformation of tetramer into monomer represented the first transition, whereas the second transition reflected the unfolding of monomer. Far-UV CD, Trp fluorescence and ANS fluorescence spectra were used to characterize the native, the intermediate and the completely denatured states of CGB lectin, obtained at 0.0 M, 5.0 M and 9.0 M urea, respectively. The intermediate state was characterized by the presence of higher secondary structures, increased ANS binding as well as increased Trp fluorescence intensity. A gradual decrease in the hemagglutination activity of CGB lectin was observed with increasing urea concentrations, showing complete loss at 4.0 M urea.
Matched MeSH terms: Protein Structure, Secondary/drug effects
There has been a spurt in the spread of microbial resistance to antibiotics due to indiscriminate use of antimicrobial agents in human medicine, agriculture, and animal husbandry. It has been realized that conventional antibiotic therapy would be less effective in the coming decades and more emphasis should be given for the development of novel antiinfective therapies. Cysteine rich peptides (CRPs) are broad-spectrum antimicrobial agents that modulate the innate immune system of different life forms such as bacteria, protozoans, fungi, plants, insects, and animals. These are also expressed in several plant tissues in response to invasion by pathogens, and play a crucial role in the regulation of plant growth and development. The present work explores the importance of CRPs as potent antimicrobial agents, which can supplement and/or replace the conventional antibiotics. Different plant parts of diverse plant species showed the presence of antimicrobial peptides (AMPs), which had significant structural and functional diversity. The plant-derived AMPs exhibited potent activity toward a range of plant and animal pathogens, protozoans, insects, and even against cancer cells. The cysteine-rich AMPs have opened new avenues for the use of plants as biofactories for the production of antimicrobials and can be considered as promising antimicrobial drugs in biotherapeutics.
We study a series of N oscillators, each coupled to its nearest neighbors, and linearly to a phonon field through the oscillator's number operator. We show that the Hamiltonian of a pair of adjacent oscillators, or a dimer, within the series of oscillators can be transformed into a form in which they are collectively coupled to the phonon field as a composite unit. In the weak coupling and rotating-wave approximation, the system behaves effectively as the trilinear boson model in the one excitation subspace of the dimer subsystem. The reduced dynamics of the one excitation subspace of the dimer subsystem coupled weakly to a phonon bath is similar to that of a two-level system, with a metastable state against the vacuum. The decay constant of the subsystem is proportional to the dephasing rate of the individual oscillator in a phonon bath, attenuated by a factor that depends on site asymmetry, intersite coupling, and the resonance frequency between the transformed oscillator modes, or excitons. As a result of the collective effect, the excitation relaxation lifetime is prolonged over the dephasing lifetime of an individual oscillator coupled to the same bath.
This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.