• BMB Reports
• /
• v.50 no.10
• /
• pp.522-527
• /
• 2017

A large number of transcriptional activation domains (TADs) are intrinsically unstructured, meaning they are devoid of a three-dimensional structure. The fact that these TADs are transcriptionally active without forming a 3-D structure raises the question of what features in these domains enable them to function. One of two TADs in human glucocorticoid receptor (hGR) is located at its N-terminus and is responsible for ~70% of the transcriptional activity of hGR. This 58-residue intrinsically-disordered TAD, named tau1c in an earlier study, was shown to form three helices under trifluoroethanol, which might be important for its activity. We carried out heteronuclear multi-dimensional NMR experiments on hGR tau1c in a more physiological aqueous buffer solution and found that it forms three helices that are ~30% pre-populated. Since pre-populated helices in several TADs were shown to be key elements for transcriptional activity, the three pre-formed helices in hGR tau1c delineated in this study should be critical determinants of the transcriptional activity of hGR. The presence of pre-structured helices in hGR tau1c strongly suggests that the existence of pre-structured motifs in target-unbound TADs is a very broad phenomenon.

• Biomedical Science Letters
• /
• v.11 no.1
• /
• pp.57-61
• /
• 2005

The number of sites at which a protein can be readily cleaved by a proteolytic enzyme is greatly influenced by its three-dimensional structure. For native, properly-folded proteins both the rate of cleavage and number of sites at which cleavage takes place are usually much less than for the denatured protein. In order to compare the tertiary structure of recombinant HepG2 type glucose transporter with that of its native counterpart in the erythrocyte, the pattern of tryptic cleavage of the protein expressed in insect cell membranes was therefore examined. After 30 minutes digestion, a fragment of approximate Mr 19,000-21,000 was generated. In addition to this, there were two less intensely stained fragments of apparent Mr 28,000 and 17,000. The pattern of labelling was similar up to 2 hours of digestion. However, the fragments of Mr 19,000-21,000 and Mr 17,000 were no longer detectable after 4 hours digestion. The observation of a very similar pattern of fragments yielded by tryptic digestion of the HepG2 type transporter expressed in insect cells suggests that the recombinant protein exhibits a tertiary structure similar if not identical to that of its human counterpart. Also, the endogenous sugar transporter(s) present in Sf21 cells did not bind cytochalasin B, the potent transporter inhibitor. Therefore, the baculovirus/Spodoptera frugiperda (Sf) cell expression system could be very useful for production of large amounts of human glucose transporters, heterologously.

• Genomics & Informatics
• /
• v.20 no.4
• /
• pp.41.1-41.9
• /
• 2022

The pathogen Gallibacterium anatis has caused heavy economic losses for commercial poultry farms around the world. However, despite its importance, the functions of its hypothetical proteins (HPs) have been poorly characterized. The present study analyzed the functions and structures of HPs obtained from Gallibacterium anatis (NCTC11413) using various bioinformatics tools. Initially, all the functions of HPs were predicted using the VICMpred tool, and the physicochemical properties of the identified virulence proteins were then analyzed using Expasy's ProtParam server. A virulence protein (WP_013745346.1) that can act as a potential drug target was further analyzed for its secondary structure, followed by homology modeling and three-dimensional (3D) structure determination using the Swiss-Model and Phyre2 servers. The quality assessment and validation of the 3D model were conducted using ERRAT, Verify3D, and PROCHECK programs. The functional and phylogenetic analysis was conducted using ProFunc, STRING, KEGG servers, and MEGA software. The bioinformatics analysis revealed 201 HPs related to cellular processes (n = 119), metabolism (n = 61), virulence (n = 11), and information/storage molecules (n = 10). Among the virulence proteins, three were detected as drug targets and six as vaccine targets. The characterized virulence protein WP_013745346.1 is proven to be stable, a drug target, and an enzyme related to the citrate cycle in the present pathogen. This enzyme was also found to facilitate other metabolic pathways, the biosynthesis of secondary metabolites, and the biosynthesis of amino acids.

• Genomics & Informatics
• /
• v.15 no.4
• /
• pp.142-146
• /
• 2017

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.

• Journal of the Korean Magnetic Resonance Society
• /
• v.3 no.2
• /
• pp.100-108
• /
• 1999

Cop protein is the transcription repressor protein in rolling circle replication plasmid. With antisense RNA, Cop protein controls the copy number of plasmid. Cop family proteins have been found in various plasmids. Among Cop family proteins, Cop studied in this paper consists of 55 amino acids (Mw. 6,400), and was known to have trimer structure. Since no structural facts are elucidated, we have carried out preliminary experiments aimed at the elucidation of its three dimensional structure. The secondary structure of Cop is studied by CD and NMR. To solve the aggregation of Cop at high concentration, we tested various detergents and salts. The addition of detergents and salts could not solve the aggregation problem. However, we found that concentration is important in solving the aggregation problem. We knew that 0.18mM in 50mM potassium phosphate without any other ingredients is maximum concentration not to aggregate. Wa also investigated the pH dependence of Cop protein, and knew that Cop protein is more stable in acid state. At various temperatures, 15N-1H HSQC spectra were measured in order to find the optimal experimental condition. To enhance the peak resolution, 3D NOESY-HSQC spectrum is acquired. Since there are NOE peaks in the NH-NH region, we knew that Cop protein has $\alpha$-helical content, which was also confirmed by CD.

• Journal of Integrative Natural Science
• /
• v.9 no.2
• /
• pp.136-143
• /
• 2016

KiSS1-derived peptide receptor, a GPCR protein, binds with the hormone kiss peptin. They are important in the neuroendocrine regulation of reproduction and in the secretion of gonadotrophin-releasing hormone. Thus, analysing the structural features of the receptor becomes important. However, the three dimensional structure of the protein is unavailable. Hence in this study, we have performed the homology modelling of KiSS1-derived peptide receptor with 5 different templates. 30 models were constructed using two platforms - Easymodeller and ITasser. The optimal models were chosen based on the model validation. Two models were selected after validation. The developed models could provide useful for analysing the structural features of KiSS1-derived peptide receptor and their pathophysiological role in various disorders related to them.

• Journal of the Korean Magnetic Resonance Society
• /
• v.20 no.1
• /
• pp.17-21
• /
• 2016

Periostin, a component of extracellular matrix (ECM) protein, is produced and secreted by the fibroblasts that are involved in chronic allergic inflammation diseases and various types of human cancers. Periostin protein is composed of multiple domains including four FAS1 domains which play important roles in cell adhesion and tumor metastasis by interacting with integrins. In spite of their important biological role, the structural information of periosin FAS1 domains was not revealed yet. Recently we systemically prepared various constructs of the FAS1 domains and tried to express them in E. coli. Of them, only single FAS1-II and -IV domains were highly soluble. Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies revealed that the FAS1-IV domain might be suitable for three-dimensional structure determination using NMR spectroscopy.

• Journal of Integrative Natural Science
• /
• v.9 no.4
• /
• pp.234-240
• /
• 2016

Bradykinin receptor B2, a GPCR protein, binds with the inflammatory mediator hormone bradkynin. It plays an important role in cross-talk between the renin-angiotensin system (RAS) and the kinin-kallikrein system (KKS). Also, it is involved in many processes including vasodilation, edema, smooth muscle spasm and pain fiber stimulation. Hence, studuying the structural features of the receptor becomes important. But the unavailability of the three dimensional structure of the protein makes the analysis difficult. Hence we have performed the homology modelling of Bradykinin receptor B2 with 5 different templates. 25 different homology models were constructed. Two best models were selected based on the model validation. The developed models could be helpful in analysing the structural features of Bradykinin receptor B2 and in pathophysiology of various disorders related to them.

• Journal of Integrative Natural Science
• /
• v.10 no.3
• /
• pp.119-124
• /
• 2017

Glucagon-like peptide 2 receptor, a GPCR, binds with the glucagon-like peptide, GLP-2 and regulates various metabolic functions in the gastrointestinal tract. It plays an important role in the nutrient homeostasis related to nutrient assimilation by regulating mucosal epithelium. GLP-2 receptor affects the cellular response to external injury, by controlling the intestinal crypt cell proliferation. As they are therapeutically attractive towards diseases related with the gastrointestinal tract, it becomes essential to analyse their structural features to study the pathophysiology of the diseases. As the three dimensional structure of the protein is not available, in this study, we have performed the homology modelling of the receptor based on single- and multiple template modeling. The models were subjected to model validation and a reliable model based on the validation statistics was identified. The predicted model could be useful in studying the structural features of GLP-2 receptor and their role in various diseases related to them.

• Applied Biological Chemistry
• /
• v.41 no.8
• /
• pp.572-577
• /
• 1998

The solution conformation of the human insulin precursor, preproinsulin, is described in terms of NMR spectral data. NMR experiments were performed on preproinsulin, whose structure was compared with the NMR structure of native human insulin. Despite the presence of the C-peptide and/or the signal peptide, secondary structure analyses indicate that the native structures of the A and B chains are well conserved even in preproinsulin. The observed relative robustness of the native structure in precursor forms permits further protein engineering experiments where the C-peptide or N-terminal signal sequence can be altered for the purpose of increasing expression or purification yields when producing recombinant human insulin.