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1.
RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain 1H, 15N, 13C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts. 相似文献
2.
Romain Navarro Olivier Bornet Laetitia Houot Roland Lloubes Françoise Guerlesquin Matthieu Nouailler 《Biomolecular NMR assignments》2016,10(2):311-313
Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIIICTX) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123–H128), 97 % of backbone 1H, 15N and 13C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689). 相似文献
3.
Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the 1H, 15N, and 13C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening. 相似文献
4.
Juanjuan Yang Yindi Liu Zhao Liu Chun Meng Donghai Lin 《Biomolecular NMR assignments》2017,11(2):175-179
Small protein B (SmpB) is an essential molecule in trans-translation which is a universal biological pathway for protein synthesis in bacteria. Trans-translation can release stalled ribosomes from defective mRNAs and target tag-protein fragments for degradation, and then restart protein synthesis. The SmpB-tmRNA complex coordinating with other components of the trans-translation system, plays vital roles in Mycobacterium tuberculosis under both stress conditions and non-replicating conditions. Thus, elucidation of molecular details and dynamic properties of the SmpB-tmRNA interaction is a crucial step towards effectively blocking trans-translation process to shorten the duration of tuberculosis treatment. Here, we report resonance assignments for 1H, 13C and 15N of M. tuberculosis SmpB (MtSmpB, spanning residues 4–133) protein determined by a suite of 2D/3D heteronuclear NMR experiments along with predicted the secondary structure. 相似文献
5.
Biling Huang Jinglin Fu Chenyun Guo Xueji Wu Donghai Lin Xinli Liao 《Biomolecular NMR assignments》2016,10(2):321-324
Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsACTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsACTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the 1H, 15N, 13C resonance assignments of MtRpsACTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsACTD_S1 and tmRNA, RNA or POA. 相似文献
6.
David P. Bowles Alexandar L. Hansen Calvin A. Rhoads Sidharth Mohan Chunhua Yuan Thomas J. Magliery 《Biomolecular NMR assignments》2018,12(2):291-295
Er-23 is a small, 51 amino acid, disulfide-rich pheromone protein used for cell signaling by Euplotes raikovi. Ten of the 51 amino acids are cysteine, allowing up to five disulfide bonds. Previous NMR work with Er-23 utilized homologously expressed protein, prohibiting isotopic labeling, and consequently the chemical shift assignments were incomplete. We have expressed uniformly 15N and 13C-labeled Er-23 in an E. coli expression system. Here we report the full backbone and side chain resonance assignments for recombinant Er-23. 相似文献
7.
8.
Tao Li Shuangli Li Chunjie Liang Jiang Zhu Maili Liu Yunhuang Yang 《Biomolecular NMR assignments》2018,12(2):249-252
The nuclear transport factor 2 (NTF2) like superfamily includes members of the NTF2 family, delta-5-3-ketosteroid isomerases, and the beta subunit of ring hydroxygenases. This family plays important roles in both eukaryotic and prokaryotic cells, and is taken as a classic example of divergent evolution because proteins in this family exhibit diverse biological functions, although share common structural features. We cloned the gene RHE_RS02845 encoding a predicted NTF2-like domain-containing protein in Rhizobium etli, and prepared U-13C/15N-labeled protein samples for its three-dimensional NMR structural determination. Here, chemical shift assignments for both backbone and side-chain atoms are reported, which is prerequisite for further structural calculation and functional research using NMR spectroscopy. 相似文献
9.
Asmita D. Pawar Deepshikha Verma Rajeev Raman Yogendra Sharma Kandala V. R. Chary 《Biomolecular NMR assignments》2017,11(2):203-206
The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell–cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named ‘Arig’) from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as ‘Arig2’. The 13C/15N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [15N-1H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete 1H, 13C and 15N NMR resonance assignments of Arig2. 相似文献
10.
One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called Nasca (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), Nasca extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that Nasca assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by Nasca have backbone RMSD 0.8–1.5 Å from the reference structures determined by traditional NMR approaches. 相似文献
11.
Andreas Ioannis Karsisiotis Oliver M. Deacon Colin Macdonald Tharin M. A. Blumenschein Geoffrey R. Moore Jonathan A. R. Worrall 《Biomolecular NMR assignments》2017,11(2):165-168
Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin. In the present study, we report backbone 1H, 13C, 15N resonance assignments of acid-denatured human cytochrome c in the aprotic solvent dimethylsulfoxide. These have led to the assignment of a reference 2D 1H-15N HSQC spectrum in which out of the 99 non-proline residues 87% of the backbone amides are assigned. These assignments are being used in an interrupted H/D exchange strategy to map the binding site of cardiolipin on human cytochrome c. 相似文献
12.
We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 + disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 + diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 + / alm1::ura4 + diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe. 相似文献
13.
Streptococcus parasanguinis is a primary colonizer of tooth surfaces in the oral cavity. Amylase-binding protein A (AbpA) from S. parasanguinis is responsible for the recruitment of salivary amylase to bacterial surface, which plays an important role in the development of oral biofilms. Here, we describe the essentially complete NMR assignments for AbpA. 相似文献
14.
Deepshikha Verma Alok Bhattacharya Kandala V. R. Chary 《Biomolecular NMR assignments》2016,10(1):67-70
We report almost complete sequence specific 1H, 13C and 15N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization. 相似文献
15.
Lihua Hou Lin Guo Chunling Wang Cong Wang 《Journal of industrial microbiology & biotechnology》2016,43(8):1131-1138
The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae. 相似文献
16.
Scott E. Sattler Jaswinder Singh Eric J. Haas Lining Guo Gautam Sarath Jeffrey F. Pedersen 《Molecular breeding : new strategies in plant improvement》2009,24(4):349-359
The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy a ; wx a ) or presence (waxy b ; wx b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx a . SEM showed that endosperm architecture was very similar in both wx a and wx b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx a and the wx b alleles to aid in molecular breeding of low amylose sorghum. 相似文献
17.
Jeremias C. Zill Zhihui He Marcus Tank Bryan H. Ferlez Daniel P. Canniffe Yigal Lahav Peter Bellstedt A. Alia Igor Schapiro John H. Golbeck Donald A. Bryant Jörg Matysik 《Photosynthesis research》2018,137(2):295-305
Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs. 相似文献
18.
Deepshikha Verma Mutyala Sakuntala Aruna Murmu Alok Bhattacharya Kandala V. R. Chary 《Biomolecular NMR assignments》2017,11(1):63-67
We report almost complete sequence specific 1H, 13C and 15N NMR assignments of an unusual Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) in its apo form as a prelude to its structural and functional characterization. 相似文献
19.
Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam SS (SS) and flam MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam py + (P) carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco +. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam SS , flam MS considerably differ from flam py + (P), and flamenco + in the structure of DIP1. Strains flam SS and flam MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam SS and flam MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco + and flam py + (P), the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome. 相似文献
20.
Meng Zhang Xiao-Wei Yu G. V. T. Swapna Gaohua Liu Rong Xiao Yan Xu Gaetano T. Montelione 《Biomolecular NMR assignments》2018,12(1):63-68
Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform 2H,13C,15N-enrichment and Ile-δ1, Leu, and Val 13CH3 methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues ?29 and ?28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone 1H, 15N, and 13C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL. 相似文献