• Bulletin of the Korean Chemical Society
• /
• v.31 no.9
• /
• pp.2581-2587
• /
• 2010

Nitration of tyrosine and tryptophan residues is common in cells under nitrative stress. However, physiological consequences of protein nitration are not well characterized on a molecular level due to limited availability of the 3D structures of nitrated proteins. Molecular dynamics (MD) simulation can be an alternative tool to probe the structural perturbations induced by nitration. In this study we developed molecular mechanics parameters for 3-nitrotyrosine (NIY) and 6-nitrotryptophan (NIW) that are compatible with the AMBER-99 force field. Partial atomic charges were derived by using a multi-conformational restrained electrostatic potential (RESP) methodology that included the geometry optimized structures of both $\alpha$- and $\beta$-conformers of a capped tripeptide ACE-NIY-NME or ACE-NIW-NME. Force constants for bonds and angles were adopted from the generalized AMBER force field. Torsional force constants for the proper dihedral C-C-N-O and improper dihedral C-O-N-O of the nitro group in NIY were determined by fitting the torsional energy profiles obtained from quantum mechanical (QM) geometry optimization with those from molecular mechanical (MM) energy minimization. Force field parameters obtained for NIY were transferable to NIW so that they reproduced the QM torsional energy profiles of ACE-NIW-NME accurately. Moreover, the QM optimized structures of the tripeptides containing NIY and NIW were almost identical to the corresponding structures obtained from MM energy minimization, attesting the validity of the current parameter set. Molecular dynamics simulations of thioredoxin nitrated at the single tyrosine and tryptophan yielded well-behaved trajectories suggesting that the parameters are suitable for molecular dynamics simulations of a nitrated protein.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.6
• /
• pp.1124-1131
• /
• 2016

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive^™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

• Bulletin of the Korean Chemical Society
• /
• v.33 no.11
• /
• pp.3597-3600
• /
• 2012

Thrombin binding aptamter (TBA-15) is a 15-mer guanine-rich oligonucleotide. This DNA apamer specifically binds to the thrombin protein involved in blood coagulation. Using extensive umbrella sampling molecular dynamics simulation method at all atom level, we investigated the potential of mean force (PMF) upon pulling the DNA aptamer from the binding mode of aptamer/thrombin complex. From this calculation, the free energy cost for a full dissociation of this aptamer/protein complex is 17 kcal/mol, indicating a substantial binding affinity of TBA-15. Interestingly, this PMF reveals noticeable plateau regions along the pulling coordinate. Possible structural changes of this complex in the plateau were investigated in details.

• Journal of the Korean Magnetic Resonance Society
• /
• v.18 no.1
• /
• pp.5-9
• /
• 2014

In last three decades, RNA molecules have been revealed to play the central roles in many cellular processes. Structural understanding of RNA molecules is essential to interpret their functions, and many biophysical techniques have been adopted for this purpose. NMR spectroscopy is a powerful tool to study structures and dynamics of RNA molecules, and it has been further applied to study tertiary interactions between RNA molecules, RNA-protein, and RNA-small molecules. This short article accounts for the general methods for NMR sample preparations, and also partially covers the resonance assignments of structured RNA molecules.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.12
• /
• pp.7473-7482
• /
• 2013

The G-protein coupled receptor 87 (GPR87) is a recently discovered orphan GPCR which means that the search of their endogenous ligands has been a novel challenge. GPR87 has been shown to be overexpressed in squamous cell carcinomas (SCCs) or adenocarcinomas in lungs and bladder. The 3D structure of GPR87 was here modeled using two templates (2VT4 and 2ZIY) by a threading method. Functional assignment of GPR87 by SVM revealed that along with transporter activity, various novel functions were predicted. The 3D structure was further validated by comparison with structural features of the templates through Verify-3D, ProSA and ERRAT for determining correct stereochemical parameters. The resulting model was evaluated by Ramachandran plot and good 3D structure compatibility was evidenced by DOPE score. Molecular dynamics simulation and solvation of protein were studied through explicit spherical boundaries with a harmonic restraint membrane water system. A DRY-motif (Asp-Arg-Tyr sequence) was found at the end of transmembrane helix3, where GPCR binds and thus activation of signals is transduced. In a search for better inhibitors of GPR87, in silico modification of some substrate ligands was carried out to form polar interactions with Arg115 and Lys296. Thus, this study provides early insights into the structure of a major drug target for SCCs.

• Genomics & Informatics
• /
• v.21 no.3
• /
• pp.32.1-32.11
• /
• 2023

Resistance to anti-tuberculosis drugs, especially ethambutol (EMB), has been widely reported worldwide. EMB resistance is caused by mutations in the embB gene, which encodes the arabinosyl transferase enzyme. This study aimed to detect mutations in the embB gene of Mycobacterium tuberculosis from Papua and to evaluate their impact on the effectiveness of EMB. We analyzed 20 samples of M. tuberculosis culture that had undergone whole-genome sequencing, of which 19 samples were of sufficient quality for further bioinformatics analysis. Mutation analysis was performed using TBProfiler, which identified M306L, M306V, D1024N, and E378A mutations. In sample TB035, the M306L mutation was present along with E378A. The binding affinity of EMB to arabinosyl transferase was calculated using AutoDock Vina. The molecular docking results revealed that all mutants demonstrated an increased binding affinity to EMB compared to the native protein (-0.948 kcal/mol). The presence of the M306L mutation, when coexisting with E378A, resulted in a slight increase in binding affinity compared to the M306L mutation alone. The molecular dynamics simulation results indicated that the M306L, M306L + E378A, M306V, and E378A mutants decreased protein stability. Conversely, the D1024N mutant exhibited stability comparable to the native protein. In conclusion, this study suggests that the M306L, M306L + E378A, M306V, and E378A mutations may contribute to EMB resistance, while the D1024N mutation may be consistent with continued susceptibility to EMB.

• Journal of the Korean Magnetic Resonance Society
• /
• v.14 no.1
• /
• pp.45-54
• /
• 2010

HP1423 (Y1423_HELPY) is a conserved hypothetical protein from H. pylori strain 26695. However, Sequence Blast result indicates that HP1423 belongs to S4 (PF01479) superfamily. According to Pfam database, the S4 domain is a small domain consisting of 60-65 amino acid residues, that probably mediates binding to RNA. In this study, we report the sequence-specific backbone resonance assignment of HP1423, which has 84 amino acid residues. We could assign unambiguously about 88% of all $^{1}H_{N}$, $^{15}N$, $^{13}C_{\alpha}$, $^{13}C_{\beta}$ and $^{13}C=O$ resonances. We could not detect the resonances from residues 15-20, and disappearance of these peaks seems to be related with the intermediate-conformational exchange. These assigned NMR peaks of HP1423 can be used for studying the role of protein dynamics in millisecond timescale, and Protein-RNA binding.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.11
• /
• pp.2647-2650
• /
• 2009

Cold shock proteins (Csps) are a subgroup of the cold-induced proteins on reduction of the growth temperature below the physiological temperature. They preferentially bind to single-stranded nucleic acids to translational regulation via RNA chaperoning. Csp plays important role in cold adaptations for the psychrophilic microorganism. Recently, Cold shock protein from psychrophilic bacteria, Colwellia psychrerythraea (CpCsp) has been identified. Three dimensional structures of a number of Csps from various microorganisms have been solved by NMR spectroscopy or X-ray crystallography, but structures of psychrophilic Csps were not studied yet. Therefore, cloning and purification protocols for further structural study of psychrophilic Csp have been optimized in this study. CpCsp was expressed in E. coli with pET-11a vector system and purified by ion exchange, size exclusion, and reverse phase chromatography. Expression and purification of CpCsp in M9 minimal media was carried out and $^{15}N$-labeled proteins with high purity over 90% was obtained. Further study will be carried out to investigate the tertiary structure and dynamics of CpCsp.

• Journal of the Korean Magnetic Resonance Society
• /
• v.18 no.1
• /
• pp.24-29
• /
• 2014

Atomistic molecular dynamics (MD) simulation has become mature enabling close approximation of the real behaviors of biomolecules. In biomolecular NMR field, atomistic MD simulation coupled with generalized implicit solvent model (GBIS) has contributed to improving the qualities of NMR structures in the refinement stage with experimental restraints. Here all-atom force fields play important roles in defining the optimal positions between atoms and angles, resulting in more precise and accurate structures. Despite successful applications in refining NMR structure, however, the research that has studied the influence of force fields in GBIS is limited. In this study, we compared the qualities of NMR structures of two model proteins, ubiquitin and GB1, under a series of AMBER force fields-ff99SB, ff99SB-ILDN, ff99SB-NMR, ff12SB, and ff13-with experimental restraints. The root mean square deviations of backbone atoms and packing scores that reflect the apparent structural qualities were almost indistinguishable except ff13. Qualitative comparison of parameters, however, indicates that ff99SB-ILDN is more recommendable, at least in the cases of ubiquitin and GB1.

• Journal of Microbiology
• /
• v.39 no.3
• /
• pp.149-153
• /
• 2001

The electron transfer reaction is one of the most essential processes of life. Not only does it provide the means of transforming solar and chemical energy into a utilizable form for all living organisms, it also extends into a range of metabolic processes that support the life of a cell. Thus, it is of great interest to understand the physical basis of the rates and reduction potentials of these reactions. To identify the major determinants of reduction potentials in redox proteins, we have chosen the simplest electron transfer protein, rubredoxin, a small (52-54 residue) iron-sulfur protein family, widely distributed in bacteria and archaea. Rubredoxins can be grouped into two classes based on the correlation of their reduction potentials with the identity of residue 44; those with Ala44 (ex: Pyrococcus furiosus) have reduction potentials that are ∼50 mV higher than those with Va144 (ex: Clostridium pasteurianum). Based on the crystal structures of rubredoxins from C. pasteurianum and P. furiosus, we propose the identity of residue 44 alone determines the reduction potential by the orientation of the electric dipole moment of the peptide bond between 43 and 44. Based on 1.5 $\AA$ resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins from C. pasteurianum, the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated.