• Journal of the Korean Magnetic Resonance Society
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• v.25 no.3
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• pp.39-44
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• 2021

The CRISPR-Cas system provides adaptive immunity for bacteria and archaea against invading phages and foreign plasmids. In the Class 1 CRISPR-Cas system, multi-subunit Cas proteins assemble with crRNA to bind to DNA targets. To disarm the bacterial defense system, bacteriophages evolved anti-CRISPR (Acr) proteins that actively inhibit the host CRISPR-Cas function. Here we report the backbone resonance assignments of AcrIF7 protein that inhibits the type I-F CRISPR-Cas system of Pseudomonas aeruginosa using triple-resonance nuclear magnetic resonance spectroscopy. We employed various computational methods to predict the structure and binding interface of AcrIF7, and assessed the model with experimental data. AcrIF7 binds to Cas8f protein via flexible loop regions to inhibit target DNA binding, suggesting that conformational heterogeneity is important for the Cas-Acr interaction.

• BMB Reports
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• v.40 no.5
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• pp.839-843
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• 2007

HP0495 (Swiss-Prot ID; Y495_HELPY) is an 86-residue hypothetical protein from Helicobacter pylori strain 26695. The function of HP0495 cannot be identified based on sequence homology, and HP0495 is included in a fairly unique sequence family. Here, we report the sequencespecific backbone resonance assignments of HP0495. About 97% of all the $^1HN$, $^{15}N$, $^{13}C{\alpha}$, $^{13}C{\beta}$, and $^{13}CO$ resonances were assigned unambiguously. We could predict the secondary structure of HP0495, by analyzing the deviation of the $^{13}C{\alpha}$ and $^{13}C{\beta}$ shemical shifts from their respective random coil values. Secondary structure prediction shows that HP0495 consists of two $\alpha$-helices and four $\beta$-strands. This study is a prerequisite for determining the solution structure of HP0495 and investigating the protein-protein interaction between HP0495 and other Helicobacter pylori proteins.

• Journal of the Korean Magnetic Resonance Society
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• v.12 no.2
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• pp.65-73
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• 2008

HP1298 (Swiss-Prot ID ; P65108) is an 72-residue protein from Helicobacter pylori strain 26695. The function of HP1298 was identified as Translation initiation factor IF-l based on sequence homology, and HP1298 is included in IF-l family. Here, we report the sequence-specific backbone resonance assignments of HP1298. About 97% of all the $^{1}HN$, $^{15}N$, $^{13}C{\alpha}$, $^{13}C{\beta}$, and $^{13}CO$ resonances could be assigned unambiguously. We could predict the secondary structure of HP1298, by analyzing the deviation of the $^{13}C{\alpha}$ and $^{13}C{\beta}$ shemical shifts from their respective random coil values. Secondary structure prediction shows that HP1298 consists of six $\beta$-strands. This study is a prerequisite for determining the solution structure of HP1298 and investigating the structure-function relationship of HP1298. Assigned chemical shift can be used for the study on interaction between HP1298 and other Helicobacter pylori proteins.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.1
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• pp.1-8
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• 2020

An extracellular matrix protein, transforming growth factor-beta-induced protein (TGFBIp/βig-h3), which is induced by transforming growth factor-β in the human cornea, skin, and matrix of many connective tissues, is associated with the adhesion, migration, proliferation, and differentiation of various cells. TGFBIp contains four homologous repeat domains, known as FAS1 domains, where certain mutations have been considered to cause corneal dystrophies. In this study, backbone NMR assignments of FAS1-3/FAS1-4 tandem domain were obtained and compared with those previously known for the isolated FAS1-4 domain. The results corroborate in solution the inter-domain interaction between FAS1-3 and FAS1-4 in TGFBIp.

• Journal of the Korean Magnetic Resonance Society
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• v.17 no.1
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• pp.59-66
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• 2013

BldD, a developmental transcription factor from Streptomyces coelicolor, is a homodimeric, DNA-binding protein with 167 amino acids in each subunit. Each monomer consists of two structurally distinct domains, the N-terminal domain (BldD-NTD) responsible for DNA-binding and dimerization and the C-terminal domain (BldD-CTD). In contrast to the BldD-NTD, of which crystal structure has been solved, the BldD-CTD has been characterized neither in structure nor in function. Thus, in terms of structural genomics, structural study of the BldD-CTD has been conducted in solution, and in the present work, mainchain NMR assignments of the recombinant BldD-CTD (residues 80-167 of BldD) could be achieved by a series of heteronuclear multidimensional NMR experiments on a [$^{13}C/^{15}N$]-enriched protein sample. Finally, the secondary structure prediction by CSI and TALOS+ analysis using the assigned chemical shifts data identified a ${\beta}-{\alpha}-{\alpha}-{\beta}-{\alpha}-{\alpha}-{\alpha}$ topology of the domain. The results will provide the most fundamental data for more detailed approach to the atomic structure of the BldD-CTD, which would be essential for entire understanding of the molecular function of BldD.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.1
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• pp.49-53
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• 2015

MRA1997 is a 76-residue conserved hypothetical protein of Mycobacterium tuberculosis H37Ra, one of the most pathogenic bacterial species and the causative agent of tuberculosis. In this study, the sequence-specific backbone resonance assignment of MRA1997 was performed using NMR spectroscopy. Approximately 88.3% of the total resonances could be unambiguously assigned. By analyzing deviations of the $C{\alpha}$ and $C{\beta}$ chemical shift values, the secondary structure of MRA1997 was calculated. The result revealed that secondary structure of MRA 1997 consists of one ${\alpha}$-helix and five ${\beta}$-sheets. Our structural study will be a footstone towards the characterization of the three-dimensional structure of MRA1997.

• Journal of the Korean Magnetic Resonance Society
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• v.17 no.2
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• pp.105-110
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• 2013

HP1492 is a NifU-like protein of Helicobacter pylori (H. pylori) and plays a role as a scaffold which transfer Fe-S cluster to Fe-S proteins like Ferredoxin. To understand how to bind to iron ion or iron-sulfur cluster, HP1492 was expressed and purified in Escherichia coli (E. coli). From the NMR measurement, we could carry out the sequence specific backbone resonance assignment of HP1492. Approximately 91% of all resonances could be assigned unambiguously. By analyzing results of CSI and TALOS from NMR data, we could predict the secondary structure of HP1492, which consists of three ${\alpha}$-helices and three ${\beta}$-sheets. This study is an essential step towards the structural characterization of HP1492.

• Journal of the Korean Magnetic Resonance Society
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• v.27 no.2
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• pp.5-9
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• 2023

t ENOD40B, a plant peptide hormone, was doubly labeled with C-13 and N-15 by recombinant production in Escherichia coli. The peptide was prepared by affinity chromatography followed by protease cleavage and reverse-phase chromatography. To elucidate the mode of action against its receptor, sucrose synthase, we proceeded to assign the backbone and side-chain resonances using a set of double and triple resonance experiments. This result will be used to determine the three-dimensional structure of the peptide at its bound state as well as to observe the chemical shift changes upon binding.

• Molecules and Cells
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• v.26 no.2
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• pp.206-211
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• 2008

HI0381 of Haemophilus influenzae was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. HI0381 is a 153-residue peptidoglycan-associated outer membrane lipoprotein, and a part of the larger Tol/Pal network. Here, we report its backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments, and secondary structure predictions. About 97% of all of the $^1HN$, $^{15}N$, $^{13}CO$, $^{13}C{\alpha}$, and $^{13}C{\beta}$ resonances covering 131 non-proline residues of the 134 residue, mature protein, were clarified by sequential and specific assignments. CSI and TALOS analyses revealed that HI0381 contains five ${\alpha}$-helices and five ${\beta}$-strands. To characterize the structure of HI0381, the effects of pH and salt concentration were investigated by CD. In addition, the structural changes occurring when HI0381 was in a membranous environment were investigated by comparing its HSQC spectra and CD data in buffer and in DPC micelles; the results showed that helix ${\alpha}4$ and strand ${\beta}4$ became aligned with the membrane. We conclude that the conformation of HI0381 is affected by the membrane environment, implying that its folded state is directly related to its function.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.2
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• pp.56-60
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• 2016

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of $^1H-^{15}N$ HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.