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1.
Ann Bot ; 133(2): 287-304, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-37832038

ABSTRACT

BACKGROUND AND AIMS: HCO3- can be a major carbon resource for photosynthesis in underwater environments. Here we investigate the underlying mechanism of uptake and membrane transport of HCO3- in submerged leaves of Hygrophila difformis, a heterophyllous amphibious plant. To characterize these mechanisms, we evaluated the sensitivity of underwater photosynthesis to an external carbonic anhydrase (CA) inhibitor and an anion exchanger protein inhibitor, and we attempted to identify components of the mechanism of HCO3- utilization. METHODS: We evaluated the effects of the external CA inhibitor and anion exchanger protein inhibitor on the NaHCO3 response of photosynthetic O2 evolution in submerged leaves of H. difformis. Furthermore, we performed a comparative transcriptomic analysis between terrestrial and submerged leaves. KEY RESULTS: Photosynthesis in the submerged leaves was decreased by both the external CA inhibitor and anion exchanger protein inhibitor, but no additive effect was observed. Among upregulated genes in submerged leaves, two α-CAs, Hdα-CA1 and Hdα-CA2, and one ß-carbonic anhydrase, Hdß-CA1, were detected. Based on their putative amino acid sequences, the α-CAs are predicted to be localized in the apoplastic region. Recombinant Hdα-CA1 and Hdß-CA1 showed dominant CO2 hydration activity over HCO3- dehydration activity. CONCLUSIONS: We propose that the use of HCO3- for photosynthesis in submerged leaves of H. difformis is driven by the cooperation between an external CA, Hdα-CA1, and an unidentified HCO3- transporter.


Subject(s)
Carbonic Anhydrases , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Photosynthesis , Anions/metabolism , Plant Leaves/metabolism , Membrane Transport Proteins/metabolism , Carbon Dioxide/metabolism
2.
Protein Expr Purif ; 188: 105975, 2021 12.
Article in English | MEDLINE | ID: mdl-34536500

ABSTRACT

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme.


Subject(s)
Genome, Plant , Oryza/genetics , Plant Proteins/genetics , Seeds/genetics , Silent Mutation , Amino Acid Sequence , Calcium/chemistry , Cations, Divalent , Cloning, Molecular , Codon , Crystallization , Edible Grain , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Deletion , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Oryza/metabolism , Plant Breeding , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Seeds/metabolism , Sequence Alignment , Sequence Homology, Amino Acid
3.
Fungal Genet Biol ; 130: 82-90, 2019 09.
Article in English | MEDLINE | ID: mdl-31026589

ABSTRACT

The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.


Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Gene Targeting/methods , Ustilaginales/genetics , Base Sequence , CRISPR-Associated Protein 9/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Fungal/genetics , Fungal Proteins/genetics , Genes, Fungal/genetics , Genetic Loci , Homologous Recombination , Ribonucleoproteins/genetics , Transformation, Genetic
4.
Protein Expr Purif ; 154: 85-90, 2019 02.
Article in English | MEDLINE | ID: mdl-30291968

ABSTRACT

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5'-triphosphates.


Subject(s)
Baculoviridae , Bombyx , Tobamovirus/genetics , Viral Proteins , Animals , Bombyx/genetics , Bombyx/metabolism , Larva/genetics , Larva/metabolism , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Viral Proteins/biosynthesis , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purification
6.
Arch Biochem Biophys ; 652: 3-8, 2018 08 15.
Article in English | MEDLINE | ID: mdl-29885290

ABSTRACT

Amylomaltase (AM) catalyzes inter- and intra-molecular transglycosylation reactions of glucan to yield linear and cyclic oligosaccharide products. The functional roles of the conserved histidine at position 461 in the active site of AM from Corynebacterium glutamicum (CgAM) was investigated. H461 A/S/D/R/W were constructed, their catalytic properties were compared to the wild-type (WT). A significant decrease in transglucosylation activities was observed, especially in H461A mutant, while hydrolysis activity was barely affected. The transglucosylation factor of the H461A-CgAM was decreased by 8.6 folds. WT preferred maltotriose (G3) as substrate for disproportionation reaction, but all H461 mutants showed higher preference for maltose (G2). Using G3 substrate, kcat/Km values of H461 mutated CgAMs were 40-64 folds lower, while the Km values were twice higher than those of WT. All mutants could not produce large-ring cyclodextrin (LR-CD) product. The heat capacity profile indicated that WT had higher thermal stability than H461A. The X-ray structure of WT showed two H-bonds between H461 and heptasaccharide analog at subsite +1, while no such bonding was observed from the model structure of H461A. The importance of H461 on substrate binding with CgAM was evidenced. We are the first to mutate an active site histidine in AM to explore its function.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/enzymology , Glycogen Debranching Enzyme System/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Calorimetry, Differential Scanning , Catalysis , Catalytic Domain , Circular Dichroism , Crystallography, X-Ray , Enzyme Stability , Glycogen Debranching Enzyme System/chemistry , Glycogen Debranching Enzyme System/genetics , Glycosylation , Hydrogen Bonding , Kinetics , Mutation , Protein Structure, Secondary , Substrate Specificity
7.
Bioorg Med Chem ; 26(8): 1929-1938, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29510947

ABSTRACT

19F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments.


Subject(s)
Adenosine Triphosphate/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Small Molecule Libraries/metabolism , Adenosine Triphosphate/chemistry , Binding Sites , Calorimetry , Drug Design , Fluorine/chemistry , Humans , Magnetic Resonance Spectroscopy , Mitogen-Activated Protein Kinase 1/chemistry , Molecular Docking Simulation , Protein Structure, Tertiary , Small Molecule Libraries/chemistry , Surface Plasmon Resonance
8.
Proc Natl Acad Sci U S A ; 111(33): E3486-95, 2014 Aug 19.
Article in English | MEDLINE | ID: mdl-25092327

ABSTRACT

The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.


Subject(s)
Genes, Viral , Immune Evasion/genetics , Mosaic Viruses/immunology , Solanum lycopersicum/virology , Alleles , Molecular Dynamics Simulation , Mosaic Viruses/genetics , Mosaic Viruses/physiology , Virus Replication
9.
Plant Cell ; 25(5): 1709-25, 2013 May.
Article in English | MEDLINE | ID: mdl-23715469

ABSTRACT

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cyclopentanes/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Oxylipins/pharmacology , Plant Proteins/genetics , Plant Roots/genetics , Adaptation, Physiological/genetics , Amino Acid Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Profiling , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Oligonucleotide Array Sequence Analysis , Oryza/genetics , Oryza/growth & development , Oryza/metabolism , Plant Growth Regulators/pharmacology , Plant Proteins/metabolism , Plant Roots/growth & development , Plant Roots/metabolism , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Salinity , Sequence Homology, Amino Acid , Sodium Chloride/pharmacology , Two-Hybrid System Techniques
10.
Sci Rep ; 14(1): 6778, 2024 03 21.
Article in English | MEDLINE | ID: mdl-38514802

ABSTRACT

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.


Subject(s)
Glucose , Hydrolases , Plant Breeding , Indoleacetic Acids/chemistry , Indoles , Edible Grain
11.
J Virol ; 86(14): 7565-76, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22573863

ABSTRACT

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/ß domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded ß-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.


Subject(s)
RNA Helicases/chemistry , Tobamovirus/enzymology , Amino Acid Sequence , Binding Sites , GTP Phosphohydrolases/chemistry , Models, Molecular , Mutation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Helicases/genetics , RNA Helicases/metabolism , Saccharomyces cerevisiae/virology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Sequence Deletion , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism
12.
PLoS Pathog ; 7(12): e1002409, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22174675

ABSTRACT

Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions.


Subject(s)
GTP-Binding Proteins/metabolism , Host-Parasite Interactions/genetics , Nicotiana/virology , Plant Proteins/metabolism , Tobacco Mosaic Virus/metabolism , Viral Proteins/metabolism , Cell Line , Chromatography, Liquid , Plant Proteins/genetics , Plants, Genetically Modified , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tandem Mass Spectrometry , Viral Proteins/genetics , Virus Replication/genetics
13.
Protein Expr Purif ; 89(1): 1-6, 2013 May.
Article in English | MEDLINE | ID: mdl-23415925

ABSTRACT

Tm-1, the protein product of Tm-1, a semidominant resistance gene of tomato, inhibits tomato mosaic virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined. In this study, an N-terminal fragment of Tm-1 (residues 1-431) as a fusion protein containing an upstream maltose-binding protein was expressed in E. coli Rosetta (DE3) cells at 30°C and then purified. The solubility of the fusion protein was greater when the cells were cultured at 30°C than when cultured at lower or higher temperatures. The purified N-terminal Tm-1 fragment from which the maltose-binding protein tag had been removed has inhibitory activity against ToMV RNA replication.


Subject(s)
Maltose-Binding Proteins/isolation & purification , Plant Proteins/isolation & purification , Solanum lycopersicum/genetics , Disease Resistance/genetics , Gene Expression Regulation, Plant , Solanum lycopersicum/chemistry , Solanum lycopersicum/virology , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/genetics , Mosaic Viruses/genetics , Mosaic Viruses/pathogenicity , Plant Diseases/genetics , Plant Diseases/virology , Plant Proteins/genetics
14.
PLoS One ; 18(2): e0281767, 2023.
Article in English | MEDLINE | ID: mdl-36795787

ABSTRACT

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Gene Editing/methods , CRISPR-Cas Systems/genetics , Plant Cells , Temperature , Plant Breeding , Plants, Genetically Modified/genetics , Genome, Plant
15.
Protein Expr Purif ; 81(1): 89-95, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21964444

ABSTRACT

Tomato mosaic virus (genus, Tobamovirus) is a member of the alphavirus-like superfamily of positive-strand RNA viruses, which include many plant and animal viruses of agronomical and clinical importance. The RNA of alphavirus-like superfamily members encodes replication-associated proteins that contain a putative superfamily 1 helicase domain. To date, a viral three-dimensional superfamily 1 helicase structure has not been solved. For the study reported herein, we expressed tomato mosaic virus replication proteins that contain the putative helicase domain and additional upstream N-terminal residues in Escherichia coli. We found that an additional 155 residues upstream of the N-terminus of the helicase domain were necessary for stability. We developed an efficient procedure for the expression and purification of this fragment and have examined factors that affect its stability. Finally, we also showed that the stable fragment has nucleoside 5'-triphosphatase activity.


Subject(s)
RNA Helicases/chemistry , Recombinant Proteins/chemistry , Tobamovirus/enzymology , Viral Proteins/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Molecular Sequence Data , Protein Stability , Protein Structure, Tertiary , RNA Helicases/genetics , RNA Helicases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tobamovirus/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
16.
Biochemistry ; 50(15): 3116-26, 2011 Apr 19.
Article in English | MEDLINE | ID: mdl-21388230

ABSTRACT

To understand the stabilization, folding, and functional mechanisms of proteins, it is very important to understand the structural and thermodynamic properties of the molten globule state. In this study, the global structure of the acid molten globule state, which we call MG1, of horse cytochrome c at low pH and high salt concentrations was evaluated by solution X-ray scattering (SXS), dynamic light scattering, and circular dichroism measurements. MG1 was globular and slightly (3%) larger than the native state, N. Calorimetric methods, such as differential scanning calorimetry and isothermal acid-titration calorimetry, were used to evaluate the thermodynamic parameters in the transitions of N to MG1 and MG1 to denatured state D of horse cytochrome c. The heat capacity change, ΔC(p), in the N-to-MG1 transition was determined to be 2.56 kJ K(-1) mol(-1), indicating the increase in the level of hydration in the MG1 state. Moreover, the intermediate state on the thermal N-to-D transition of horse cytochrome c at pH 4 under low-salt conditions showed the same structural and thermodynamic properties of the MG1 state in both SXS and calorimetric measurements. The Gibbs free energy changes (ΔG) for the N-to-MG1 and N-to-D transitions at 15 °C were 10.9 and 42.2 kJ mol(-1), respectively.


Subject(s)
Cytochromes c/chemistry , Horses , Animals , Calorimetry, Differential Scanning , Circular Dichroism , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Osmolar Concentration , Protein Conformation/drug effects , Salts/pharmacology , Scattering, Small Angle , Solutions , Temperature , Thermodynamics , X-Ray Diffraction
17.
J Mol Recognit ; 24(2): 275-82, 2011.
Article in English | MEDLINE | ID: mdl-21360613

ABSTRACT

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.


Subject(s)
Calorimetry/methods , Gibberellins/metabolism , Molecular Dynamics Simulation , Plant Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Gibberellins/chemistry , Hydrogen Bonding , Kinetics , Plant Proteins/chemistry , Protein Binding , Thermodynamics
18.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 12): 1649-52, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-22139189

ABSTRACT

Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.


Subject(s)
RNA Helicases/chemistry , Tobamovirus/enzymology , Crystallization , Crystallography, X-Ray
19.
Nature ; 437(7059): 693-8, 2005 Sep 29.
Article in English | MEDLINE | ID: mdl-16193045

ABSTRACT

Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice.


Subject(s)
Gibberellins/metabolism , Oryza/genetics , Oryza/metabolism , Plant Proteins/metabolism , Protein Binding , Amino Acid Sequence , Binding, Competitive , Cloning, Molecular , Epistasis, Genetic , Genes, Plant/genetics , Gibberellins/pharmacology , Molecular Sequence Data , Mutation/genetics , Oryza/drug effects , Phenotype , Plant Proteins/chemistry , Plant Proteins/genetics , Solubility , Sterol Esterase/chemistry
20.
J Med Chem ; 64(19): 14299-14310, 2021 10 14.
Article in English | MEDLINE | ID: mdl-34582207

ABSTRACT

Fragment-based screening using 19F NMR (19F-FS) is an efficient method for exploring seed and lead compounds for drug discovery. Here, we demonstrate the utility and merits of using 19F-FS for methionine γ-lyase-binding fragments, together with a 19F NMR-based competition and mutation assay, as well as enzymatic and in silico methods. 19F NMR-based assays provided useful information on binding between 19F-FS hit fragments and target proteins. Although the 19F-FS and enzymatic assay were weakly correlated, they show that the 19F-FS hit fragments contained compounds with inhibitory activity. Furthermore, we found that in silico calculations partially account for the differences in activity levels between the 19F-FS hits as per NMR analysis. A comprehensive approach combining the 19F-FS and other methods not only identified fragment hits but also distinguished structural differences in chemical groups with diverse activity levels.


Subject(s)
Carbon-Sulfur Lyases/antagonists & inhibitors , Enzyme Assays , Enzyme Inhibitors/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Small Molecule Libraries/chemistry , Computer Simulation , Enzyme Inhibitors/pharmacology , Fluorine , Ligands , Small Molecule Libraries/pharmacology
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