Protein Data Bank ID Research Articles

Comparative sequence- and structure-inspired drug design for PilF protein of Neisseria meningitidis.

Serogroup A of Neisseria meningitidis is the organism responsible for causing epidemic diseases in developing countries by a pilus-mediated adhesion to human brain endothelial cells. Type IV pilus assembly protein (PilF) associated with bacterial adhesion, aggregation, invasion, host cell signaling, surface motility, and natural transformation can be considered as a candidate for effective anti-meningococcal drug development. Since the crystal structure of PilF was not available, in the present study, it was modeled after the Z2491 strain (CAM09255.1) using crystal structure of chain A of Vibrio cholerae putative Ntpase EpsE (Protein Data Bank (PDB) ID: 1P9R) and then we based this analysis on sequence comparisons and structural similarity using in silico methods and docking processes, to design a suitable inhibitor molecule. The ligand 3-{(4S)-5-{[(1R)-1-cyclohexylethyl]amino}-4-[(5S)-5-(prop-2-en-1-yl) cyclopent-1-en-1-yl]-1,4-dihydro-7H-pyrrolo[2,3-d] pyrimidin-7-yl}-1,2-dideoxy-b-L-erythro-hex-1-en-3-ulofuranosyl binds to the protein with a binding energy of −8.10 kcal and showed a drug likeness of 0.952 with no predicted health hazard. It can be utilized as a potent inhibitor of N. meningitidis pilus-mediated adhesion to human brain endothelial cells preventing meningeal colonization.

Synthesis and Molecular Docking Studies of Novel 2‐Phenyl‐4‐Substituted Oxazole Derivatives as Potential Anti‐cancer Agents

A novel series of 2,4‐disubstituted oxazole derivatives were synthesized, screened for their anti‐tumor activity against three cell lines MCF‐7, TK‐10, and UACC‐62. Molecular docking study was carried out against epidermal growth factor receptor. A new series of 2‐phenyl‐4‐substituted oxazole derivatives were synthesized. A series of chiral α‐amino acid derivatives 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 were synthesized by coupling various l‐acylated amino acid azide 3. The synthesized compounds were tested for their in vitro antitumor activity against MCF‐7, TK‐10, and UACC‐62 cell lines. Compound 6 exhibited the strongest inhibitory activity against TK cell lines, while compound 12 showed the highest activity against MCF‐7 cell lines. Compound 14 was the most active against UACC‐62 cell lines. Furthermore, a molecular docking study of the most active compounds was carried out using epidermal growth factor receptor X‐ray 3D structure (protein data bank ID 1 M17). Docking results revealed that compound 6 showed the highest binding energy of ΔG = −78.17 Kcal/mol.

Structure and reaction mechanism of a novel enone reductase.

Recently, a novel gut-bacterial fatty acid metabolism, saturation of polyunsaturated fatty acid, that modifies fatty acid composition of the host and is expected to improve our health by altering lipid metabolism related to the onset of metabolic syndrome, was discovered in Lactobacillusplantarum AKU 1009a. Enzymes constituting the pathway catalyze sequential reactions of free fatty acids without CoA or acyl carrier protein. Among these enzymes, CLA-ER was identified as an enone reductase that can saturate the C=C bond in the 10-oxo-trans-11-octadecenoic acid (KetoB) to produce 10-oxo-octadecanoic acid (KetoC). This enzyme is the sole member of the NADH oxidase/flavin reductase family that has been identified to exert an enone reduction activity. Here, we report both the structure of holo CLA-ER with cofactor FMN and the KetoC-bound structure, which elucidate the structural basis of enone group recognition of free fatty acids and provide the unique catalytic mechanism as an enone reductase in the NADH oxidase/flavin reductase family. A 'cap' structure of CLA-ER underwent a large conformational change upon KetoC binding. The resulting binding site adopts a sandglass shape and is positively charged at one side, which is suitable to recognize a fatty acid molecule with enone group. Based on the crystal structures and enzymatic activities of several mutants, we identified C51, F126 and Y101 as the critical residues for the reaction and proposed an alternative electron transfer pathway of CLA-ER. These findings expand our understanding of the complexity of fatty acid metabolism. The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID 4QLX, 4QLY).

An extended CCR5 ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions.

C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505.

Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M.

The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho) inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO) as a template in MODELLER (v 9.10). The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9) and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial) database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC24934545 and ZINC72319544 – that showed high binding affinity among 2,829 drug analogs that bind with key active-site residues; these residues are considered for protein-ligand binding and unbinding pathways via steered molecular dynamics simulations. Arg215 in the model plays an important role in the stability of the protein-ligand complex via a hydrogen bonding interaction by aromatic-π contacts, and the ADMET (absorption, distribution, metabolism, and excretion) analysis of best leads indicate nontoxic in nature with good potential for drug development.

Role of quassinoids as potential antimalarial agents: An in silico approach.

Background:Malaria is an infection caused by mosquitoes in human beings which can be dangerous if untreated. A well known plant product, quassinoids are known to have antimalarial activity. These bioactive phytochemicals belong to the triterpene family. Quassinoids are used in the present study to act against malarial dihydrofolate reductase (Pf-DHFR), a potential antimalarial target. Nevertheless, viṣama jvara (~malaria) has been treated with the bark of Cinchona since a long time.Aim:The aim of the present experiment is to perform the protein-ligand docking for Pf- DHFR and Quassinoids and study their binding affinities.Setting and Design:The software used for the present study is the discovery studio (Accelrys 2.1), Protein Data Bank (PDB), and Chemsketch.Materials and Methods:The protein for the present study was imported from protein data bank with the PDB Id, 4dpd and was prepared for docking. The ligands used for the study are the quassinoids. They were drawn using chemsketch and the 3D structures were generated. The docking was done subsequently.Statistical Analysis Used:Molecular modeling technique was used for the protein-ligand docking analysis.Results:The docking results showed that the Quassinoids Model_1 showed the highest dock score of 40.728.Conclusion:The present study proves the promising potential of quassinoids as novel drugs against malaria. The dock results conclude that the quassinoids can be adopted as an alternative drug against malaria.

Methanol extract of wheatgrass induces G1 cell cycle arrest in a p53-dependent manner and down regulates the expression of cyclin D1 in human laryngeal cancer cells-an in vitro and in silico approach.

Background:Deregs been implicated in the malignancy of cancer. Since many years investigation on the traditional herbs has been the focus to develop novel and effective drug for cancer remedies. Wheatgrass is a medicinal plant, used in folk medicine to cure various diseases. The present study was undertaken to gain insights into antiproliferative effect of methanol extract of wheatgrass.Materials Methods:Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase assays. Cell cycle was analyzed by flow cytometry. Western blot was performed to determine the p53 and cyclin D1 levels. In silico docking interaction of the 14 active components (identified by high-performance liquid chromatography/gas chromatography-mass spectroscopy) of the methanol extract was tested with cyclin D1 (Protein Data Bank ID: 2W96) and compared with the reference cyclin D1/Cdk4 inhibitor.Results:Methanol extract of wheatgrass effectively reduced the cell viability. The cell cycle analysis showed that the extract treatment caused G1 arrest. The level of cyclin D1 was decreased, whereas p53 level was increased. Molecular docking studies revealed interaction of seven active compounds of the extract with the vital residues (Lys112/Glu141) of cyclin D1.Conclusion:These findings indicate that the methanol extract of wheatgrass inhibits human laryngeal cancer cell proliferation via cell cycle G1 arrest and p53 induction. The seven active compounds of the extract were also found to be directly involved in the inhibition of cyclin D1/Cdk4 binding, thus inhibiting the cell proliferation.

Prediction of anticancer property of bowsellic acid derivatives by quantitative structure activity relationship analysis and molecular docking study.

Context:Boswellic acid consists of a series of pentacyclic triterpene molecules that are produced by the plant Boswellia serrata. The potential applications of Bowsellic acid for treatment of cancer have been focused here.Aims:To predict the property of the bowsellic acid derivatives as anticancer compounds by various computational approaches.Materials and Methods:In this work, all total 65 derivatives of bowsellic acids from the PubChem database were considered for the study. After energy minimization of the ligands various types of molecular descriptors were computed and corresponding two-dimensional quantitative structure activity relationship (QSAR) models were obtained by taking Andrews coefficient as the dependent variable.Statistical Analysis Used:Different types of comparative analysis were used for QSAR study are multiple linear regression, partial least squares, support vector machines and artificial neural network.Results:From the study geometrical descriptors shows the highest correlation coefficient, which indicates the binding factor of the compound. To evaluate the anticancer property molecular docking study of six selected ligands based on Andrews affinity were performed with nuclear factor-kappa protein kinase (Protein Data Bank ID 4G3D), which is an established therapeutic target for cancers. Along with QSAR study and docking result, it was predicted that bowsellic acid can also be treated as a potential anticancer compound.Conclusions:Along with QSAR study and docking result, it was predicted that bowsellic acid can also be treated as a potential anticancer compound.

Metabolic pathway analysis approach: Identification of novel therapeutic target against methicillin resistant Staphylococcus aureus

Multiple Drug Resistant (MDR) bacteria are no more inhibited by the front line antibiotics due to extreme resistance. Methicillin Resistant Staphylococcus aureus (MRSA) is one of the MDR pathogens notorious for its widespread infection around the world. The high resistance acquired by MRSA needs a serious concern and efforts should be carried out for the discovery of better therapeutics. With this aim, we designed a comparison of the metabolic pathways of the pathogen, MRSA strain 252 (MRSA252) with the human host (i.e., Homo sapiens) by using well-established in silico methods. We identified several metabolic pathways unique to MRSA (i.e., absent in the human host). Furthermore, a subtractive genomics analysis approach was applied for retrieval of proteins only from the unique metabolic pathways. Subsequently, proteins of unique MRSA pathways were compared with the host proteins. As a result, we have shortlisted few unique and essential proteins that could act as drug targets against MRSA. We further assessed the druggability potential of the shortlisted targets by comparing them with the DrugBank Database (DBD). The identified drug targets could be useful for an effective drug discovery phase. We also searched the sequences of unique as well as essential enzymes from MRSA in Protein Data Bank (PDB). We shortlisted at least 12 enzymes for which there was no corresponding deposition in PDB, reflecting that their crystal structures are yet to be solved! We selected Glutamate synthase out of those 12 enzymes owing to its participation in significant metabolic pathways of the pathogen e.g., Alanine, Aspartate, Glutamate and Nitrogen metabolism and its evident suitability as drug target among other MDR bacteria e.g., Mycobacteria. Due to the unavailability of any crystal structure of Glutamate synthase in PDB, we generated the 3D structure by homology modeling. The modeled structure was validated by multiple analysis tools. The active site of Glutamate synthase was identified by not only superimposing the template structure (PDB ID: 1E0A) over each other but also by the Parallel-ProBiS algorithm. The identified active site was further validated by cross-docking the co-crystallized ligand (2-oxoglutaric acid; AKG) of PDB ID: 1LLW. It was concluded that the comparative metabolic in silico analysis together with structure-based methods provides an effective approach for the identification of novel antibiotic targets against MRSA.

The Structure of NMDA Receptor Revealed

++ ions to flow across the cell membrane through the pore. Furthermore, activated NMDA receptors also allow the entry of Ca 2+ into the cell which will then trigger a series of cellular events, such as activations of Ca 2+/ calmodulin-depedent protein kinase (CaMKII), protein kinase C (PKC) and the insertion of another type of glutamate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, into the postsynaptic membrane via exocytosis [2], so that the synaptic response to glutamate is strengthened. This is believed to be the basis of learning and memory. Dysfunctions of NMDA receptors may contribute to pain sensation and various brain disorders such as depression, schizophrenia, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease [3]. Recently, two independent studies, by Karakas and Furukawa [4] and Lee et al. [5], revealed the first insight of the crystal structure of NMDA receptor in almost complete configurations. In both studies, the NMDA receptors are heterotetrameric complexes comprised of two GluN1 and two GluN2B subunits. In order to stabilize the crystal structures, both groups used ligands, agonists, or antagonists, together with introductions of multiple mutations in the subunits and eventually obtained crystals of NMDA receptor in an inactive state. Both structures (Protein Data Bank IDs: 4PE5 and 4TLL) have resolutions better than 4A, and both show two key characteristics reminiscent of the subunit organization of AMPA receptor obtained five years ago [6]. First, the two GluN1 subunits and the two GluN2 subunits are arranged in an alternate pattern around the central pore, with twofold symmetry between the two GluN1-GluN2 heterodimers. Secondly, the receptor domains are layered. The amino terminal domain (ATD), which controls the gating of the channel and allows modulator bindings, is at the exterior end. The transmembrane domain (TMD), which provides the basis of the ion channel stays in the cell membrane. The ligand-binding domain (LBD), hosting the binding sites of ligands or their agonists and antagonists, is sandwiched between the ATD and the LBD. Going from the ATD to the LBD, the subunits in the two heterodimers swap domains,

Insilico Identification of Potent Inhibitor from Andrographis Paniculata for ESBLs

<span lang="EN-US">The study aimed at docking of TEM - 52 & modeled CTX – M -13 by ethanol extracted compounds from <em>Andrographis paniculata</em> and to predict their binding efficiency. BLAST-P was performed to retrieve suitable templates for homology-modeling using the bla CTX-M sequences obtained from Genbank. Protein Data Bank (PDB) IDs of these templates were retrieved. Models were prepared using Swiss-Model-Server and verified by Procheck and 3D programmes RAMPAGE was used to prepare Ramachandran plots. The 13 - Hexyl - oxa - cyclotridec - 10 - En- 2 - one was selected as ligand for this docking study from the data generated by Mass spectrometry analysis of methanol extracted compounds from <em>Andrographis paniculata</em>. The structure was drawn manually with aid of JME and its drug likeliness & molecular properties was calculated using Pre Admet server. Docking was performed by using docking server. Since the docking results proved that the amino acid residues Ser70,Ser 130,Ser237,Arg 276 and Thr235 of CTX-M-13 (enzyme) make important contacts with the ligand, researchers are expected to duly utilize this information for designing more potent and CTX - M inhibitors.</span>

Application of maximum entropy method to neutron protein crystallography

Hydrogen atoms or hydrogen bonds play important roles in protein functions. Neutron diffraction is very powerful tool to detect hydrogen atoms. However it is often obtained rather poor resolution data compared with X-ray data due to weak neutron source intensity and large incoherent scattering from hydrogen atoms. The maximum entropy method (MEM) is noble method to obtain high resolution electron or nuclear density distribution from even limited number of diffraction data. The MEM has been applied to not only X-ray data of small molecules but also those of proteins. For the application to neutron data, so far, only small molecules are reported. When preliminary application of the MEM to 1.1 Å resolution partially deuterated neutron protein data (Protein Data Bank ID : 4fc1) is carried out, most of hydrogen and deuterium atoms are observed (Fig. 1). Since this resolution is unusually high, it is of interest that ability of the MEM for usual or low resolution data. In this paper, effects of resolution and data quality for the MEM are examined. Large deuterated crystals are necessary for neutron experiment to reduce incoherent scattering from hydrogen atoms and to improve data resolution. However deuterated condition is not usual for biomolecules. If no deuteration is need for low resolution data by using the MEM, it would be able to observe biomolecules as they are.

Discovery and design of cyclic peptides as dengue virus inhibitors through structure-based molecular docking

ObjectiveTo find potential peptide inhibitors against the NS2B/NS3 protease of DENV which in turn, can inhibit the viral replication inside host cell. MethodsCyclic peptides were designed having combination of positively charged amino acids using ChemSketch software and were converted to 3D structures. DENV NS3 protein structure was retrieved from Protein Data Bank (PDB) using PDB Id: 2FOM. DENV NS3 and cylic peptides were docked using MOE software after structural optimization. ResultsThrough molecular docking it was revealed that most of the peptides bound deeply in the binding pocket of DENV NS2B/NS3 protease an had interactions with catalytic triad. Peptide 2 successfully blocked the catalytic triad of NS2B/NS3 protease. Peptide 1, 4 and 6 also had potential interactions with active residues of the NS2B/NS3 protease while all other peptides were in close contact with the active sites of NS2B/NS3 protease thus, these peptides can serve as a potential drug candidate to stop viral replication. ConclusionsThus, it can be concluded from the study that these peptides could serve as important inhibitors to inhibit the viral replication and need further in-vitro investigations to confirm their efficacy.

Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

Homology modelling and molecular dynamics simulations of a protein serine/threonine phosphatase stp1 in Staphylococcus aureus N315: a potential drug target

The prevalence of methicillin-resistant Staphylococcus aureus and vanomycin intermediate S. aureus infections is on the rise, globally. This poses a huge challenge due to limited therapeutic options and the limited number of bacterial-specific drug targets available for due conservation with the human host. A serine/threonine phosphatase/kinase stp1/stk1 phospho-signalling system in S. aureus, which is just beginning to be understood, has been shown to be of importance in virulence and susceptibility to glycopeptide antibiotics. In this study, 3D structure of stp1 (clinical strain of S. aureus N315) was predicted using a homology modelling tool MODELLER. The validation of the predicted model was done using various tools such as PROCHECK, ERRAT, VERIFY-3D and ProSA. Molecular dynamics (MD) study was carried out using GROMACS to refine the least energy model generated from MODELLER9v11 and it was compared with the template. The template used was the crystal structure of serine/threonine phosphatase stp1 in Streptoccocus agalactiae (Protein Data Bank ID: 2PK0) with 38% identity with the query. Various validation tools showed the quality of the model generated using MODELLER. PROCHECK predicted 100% residues in the allowed region, ERRAT with overall quality factor of 76.47, VERIFY-3D with average score of >0.2 in 81.78% of residues, WHATIF with packaging quality score of > − 5 for all residues and ProSA with Z-score of − 7.02. MD simulation of the protein showed some fluctuations in the aqueous environment and changes in the ligand binding residues after simulation. The availability of the 3D-structural information of a viable drug target in S. aureus stp1 is expected to facilitate structure–activity relationship and interactions with proteins.

Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis

Mycobacterium tuberculosis is a gram-positive bacterium causes tuberculosis in human. H37Rv strain is a pathogenic strain utilized for tuberculosis research. The cytidylate mono-phosphate (CMP) kinase of Mycobacterium tuberculosis belongs to the family nucleoside mono-phosphate kinase (NMK), this enzyme is required for the bacterial growth. Therefore, it is important to study the structural and functional features of this enzyme in the control of the disease. Hence, we developed the structural molecular model of the CMP kinase protein from Mycobacterium tuberculosis by homology modeling using the software MODELLER (9v10). Based on sequence similarity with protein of known structure (template) of Mycobacterium smegmatis (PDB ID: 3R20) was chosen from protein databank (PDB) by using BLASTp. The energy of constructed models was minimized and the qualities of the models were evaluated by PROCHECK and VERRIFY-3D. Resulted Ramachandran plot analysis showed that conformations for 100.00% of amino acids residues are within the most favored regions. A possible homologous deep cleft active site was identified in the Model using CASTp program. Amino acid composition and polarity of that protein was observed by CLC-Protein Workbench tool. Expasy's Prot-param server and CYC_REC tool were used for physiochemical and functional characterization of the protein. Studied of secondary structure of that protein was carried out by computational program, ProFunc. The structure is finally submitted in Protein Model Database. The predicted model permits initial inferences about the unexplored 3D structure of the CMP kinase and may be promote in relational designing of molecules for structure-function studies.

Potential of human γD‐crystallin for hair damage repair: insights into the mechanical properties and biocompatibility

The objective of this work was to develop a new strategy to physically 'repair' chemically damaged hair. Hence the human eye γD-crystallin, a protein from the superfamily characterized structurally by the Greek key motif, was studied. The human γD-crystallin was chosen based on the ability of proteins belonging to this superfamily to be involved in the coating of specific structures. Two crystallins were used on the study, the wild type (Protein Data Bank ID: 1HK0) and the mutant protein. The mutant form was intended to induce a strong and quick protein polymerization as well to have new possible points of anchorage to hair. The ability of both crystallins to bind to damaged hair and even penetrate into its cortex was checked by fluorescence microscopy, confocal microscopy and scanning electron microscopy. Furthermore the reinforcement of hair mechanical resistance, the potential cytotoxic/inflammatory effect of crystallins were studied in order to have a fully comprehension about the protein based formulation. Although the chemical over-bleaching treatment induced a decrease of 20% on the resistance of the hair, the crystallins which bind and penetrate the hair fibre were able to recover and even to improve its mechanical properties when compared to the virgin hair. Moreover none of the crystallins displayed a toxic effect in fibroblasts for all the range of tested concentrations upon 72 h of exposure. The active aggregation process of mutant crystallin induced an inflammatory response in fibroblasts in the first 24 h of contact, measured by the amount of released pro-inflammatory cytokine IL-6 to the medium. In contrast contact with wild type crystallin did not lead to significant inflammation. Outcome from protein formulation characterization supports the hypothesis that the γD-crystallin it is able to recover and improve the mechanical properties of chemical damaged hair. Therefore it can be considered as a very promising strengthening agent for the development of new restorative hair care products.

Reply to Campos and Muñoz: Why phosphate is a bad buffer for guanidinium chloride titrations

Liu et al. (1) alleged that they could not repeat our work on the protein BBL (Protein Data Bank ID code 1W4H) at the most basic level. Campos and Munoz now admit (2), as we explained (3), that they used a phosphate buffer that greatly changed pH during the guanidinium chloride (GdmCl) titration relative to our Mops buffer. They can now repeat our work.