Single Amino Acid Insertion Research Articles

Diverse Proteomimetic Frameworks via Rational Design of π-Stacking Peptide Tectons.

Peptide-based frameworks aim to integrate protein architecture into solid-state materials using simpler building blocks. Despite the growing number of peptide frameworks, there are few strategies to rationally engineer essential properties like pore size and shape. Designing peptide assemblies is generally hindered by the difficulty of predicting complex networks of weak intermolecular interactions. Peptides conjugated to polyaromatic groups are a unique case where assembly appears to be strongly driven by π-π interactions, suggesting that rationally adjusting the geometry of the π-stackers could create novel structures. Here, we report peptide elongation as a simple mechanism to predictably tune the angle between the π-stacking groups to produce a remarkable diversity of pore shapes and sizes, including some that are mesoporous. Notably, rapid jumps in pore size and shape can occur with just a single amino acid insertion. The geometry of the π-stacking residues also significantly influences framework structure, representing an additional dimension for tuning. Lastly, sequence identity can also indirectly modulate the π-π interactions. By correlating each of these factors with detailed crystallographic data, we find that, despite the complexity of peptide structure, the shape and polarity of the tectons are straightforward predictors of framework structure. These guidelines are expected to accelerate the development of advanced porous materials with protein-like capabilities.

Undesirable protein sequence variations in maize genes that confer resistance to fungal pathogens and insect pests

Diseases and insect pests greatly impact sustainable production in maize. Maize inbred lines have varying levels of resistance to these pathogens and insects, but little is known about the diversity of their resistance proteins. In this study, genes encoding seven proteins that are involved in resistance to insects and pathogens in maize were analyzed in 46 maize inbred lines to elucidate the differences in amino acid sequences. The proteins of interest are superlectin, maizewin, hydrolase, geranyl geranyl transferase, quinone oxidoreductase, AIL1, and defensin. The protein sequences encoded by genes for superlectin, AIL1, and defensin were found to be disrupted in some maize inbreds but were conserved in others. The characterized disruptions resulted from single amino acid changes, insertions, or deletions. While the effect of single amino acid changes is hard to predict, insertions and deletions likely disrupt protein function, increasing the susceptibility of maize plants to insects and/or diseases. Functional resistance genes can be incorporated from the identified maize inbreds into commercial hybrids to promote enhanced insect and pathogen resistance.

The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

A single amino acid insertion in LCYB2 deflects carotenoid biosynthesis in red carrot.

A single amino acid insertion in LCYB2 deflects carotenoid biosynthesis in red carrot.

Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae

ObjectivesTo analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. MethodsClinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. ResultsMolecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, four of which were novel mutational changes. ConclusionsDifferent mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.

Fitness Effects of Single Amino Acid Insertions and Deletions in TEM-1 β-Lactamase

Short insertions and deletions (InDels) are a common type of mutation found in nature and a useful source of variation in protein engineering. InDel events have important consequences in protein evolution, often opening new pathways for adaptation. However, much less is known about the effects of InDels compared to point mutations and amino acid substitutions. In particular, deep mutagenesis studies on the distribution of fitness effects of mutations have focused almost exclusively on amino acid substitutions. Here, we present a near-comprehensive analysis of the fitness effects of single amino acid InDels in TEM-1 β-lactamase. While we found InDels to be largely deleterious, partially overlapping deletion-tolerant and insertion-tolerant regions were observed throughout the protein, especially in unstructured regions and at the end of helices. The signal sequence of TEM-1 tolerated InDels more than the mature protein. Most regions of the protein tolerated insertions more than deletions, but a few regions tolerated deletions more than insertions. We examined the relationship between InDel tolerance and a variety of measures to help understand its origin. These measures included evolutionary variation in β-lactamases, secondary structure identity, tolerance to amino acid substitutions, solvent accessibility, and side-chain weighted contact number. We found secondary structure, weighted contact number, and evolutionary variation in class A beta-lactamases to be the somewhat predictive of InDel fitness effects.

Subclade 2.2.1-Specific Human Monoclonal Antibodies That Recognize an Epitope in Antigenic Site A of Influenza A(H5) Virus HA Detected between 2015 and 2018.

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144–147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144–147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144–147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.

Identification and characterization of BATF3 as a context-specific coactivator of the glucocorticoid receptor.

The ability of the glucocorticoid receptor (GR) to regulate the transcriptional output of genes relies on its interactions with transcriptional coregulators. However, which coregulators are required for GR-dependent activation is context-dependent and can be influenced by the sequence of the DNA bound by GR and by the nature of the GR isoform responsible for the regulation of a gene. Here, we screened for GR-interacting proteins for which the interaction signal differed between two GR isoforms GRα and GRγ. These isoforms diverge by a single amino acid insertion in a domain, the lever arm, which adopts DNA sequence-specific conformations. We identify Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), an AP-1 family transcription factor, as a GR coregulator whose interaction with GR is modulated by the lever arm. Further, a combination of experiments uncovered that BATF3 acts as a gene-specific coactivator of GR whose coactivator potency is influenced by the sequence of the GR binding site. Together, our findings suggest that GR isoform and the sequence of GR binding site influence the interaction of GR with BATF3, which might direct the assembly of gene-specific regulatory complexes to fine-tune the expression of individual GR target genes.

Engineering the cofactor specificity of an alcohol dehydrogenase via single mutations or insertions distal to the 2'-phosphate group of NADP(H).

There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2'-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a 'loop-grafting' approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity. Here, we further explore this approach by introducing single point mutations and single amino acid insertions at the loop insertion site. Six different mutants of AdhD were created by either converting glycine 211 to cysteine or serine or by inserting alanine, serine, glycine or cysteine between the 211 and 212 residues. Several mutants gained activity with NADP+ above the wild-type enzyme. And remarkably, it was found that all of the mutants investigated resulted in some degree of reversal of cofactor specificity in the oxidative direction. These changes were generally a result of changes in conformations of the ternary enzyme/cofactor/substrate complexes as opposed to changes in affinities or binding energies of the cofactors. This study highlights the role that amino acids which are distal to the cofactor binding pocket but are involved in substrate interactions can influence cofactor specificity in AdhD, and this strategy should translate to other AKR family members.

Phylogenetic and genome analysis of seven senecavirus A isolates in China.

Senecavirus A (SVA) is the only member of genus Senecavirus that causes vesicular lesions in pigs. We have characterized seven SVA isolates from different swine farms in Guangdong, China. The most variable isolate, CH-DL-01-2016, contained a single amino acid insertion at position 219-220 and a 16 amino acid insertion at position 250-251. The VP1 protein also had four nucleotide changes when compared to 31 other SVA VP1 sequences obtained from GenBank. These mutations have not been identified before. Phylogenetic trees demonstrated that the new SVA isolates were clustered into two different clades and shared 96.3%-97.1% similarity with US strains and 97.9%-98.3% similarity with Brazilian stains on nucleotide level, respectively. Prediction of antigenic epitope indicated that SVA VP1 protein contained both potential B-cell and potential T-cell epitopes. This report provides information about variation analysis in SVA in southern China.

765. Detection of Treatment-Resistant Infectious HIV After Genome-Directed Antiviral Endonuclease Therapy

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One newly emerging approach to cure persistent viral infections is via the use of targeted endonucleases. To date, endonucleases have been used as therapeutic agents targeting the genomes of viruses including EBV, JCV, HBV, HCV, HIV, HPV, HSV and HTLV, and have been shown to inhibit viral replication and subsequently persistence. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of a panel of four HIV pol-specific zinc finger nucleases (ZFNs) we identified a provirus encoding a treatment-resistant and infectious mutant virus that was derived from a ZFN2-mediated disruption of reverse transcriptase (RT). Although ZFN-mediated disruption of HIV protease (ZFN1), RT (ZFN2 & ZFN3) and integrase (ZFN4) coding sequences could inhibit viral replication, a RT mutant provirus was detected that produced a replication competent and ZFN2 cleavage resistant HIV. Mutant virus ZFN2(+3) contained a single amino acid insertion that introduced a Leucine-Leucine (LL) motif in the thumb domain of RT. The RT of mutant virus ZFN2(+3) likely remained functional since a LL motif is also found in the RT of several hominid, old world monkey or equine spumaviruses. In ZFN2-treated HEK293 and CD4+ SupT1 T cells, we found that up to 25% and 6% of all ZFN2 HIV pol target site mutations detected by Illumina sequencing encoded the ZFN2(+3) mutant respectively. We found that ZFN2(+3) mutant virus could replicate at levels comparable to wild type HIV in primary CD4+ T cells, but importantly remained susceptible to treatment with both NRTi and NNRTi antiretroviral inhibitors. When secondary ZFN-derived mutations were introduced into other RT or integrase domains of mutant virus ZFN2(+3), replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

Identification of an amphipathic helix within IFITM3 that promotes inhibition of influenza virus

Abstract Interferon-induced transmembrane protein 3 (IFITM3) inhibits influenza virus fusion with endosomes by altering cellular membrane properties through mechanisms that are largely uncharacterized. In order to identify regions of secondary structure within IFITM3 that may contribute to its mechanism of action, we performed structural predictions using the I-TASSER and PSSpred algorithms. Insterestingly, we identified a 10-amino acid region of likely helicity that possesses an amphipathic distribution of amino acids. Consistent with the known role of amphipathic helices in altering membrane properties, we observed that cells expressing a helix-deleted mutant of IFITM3 were infected with influenza virus at a 4-fold higher rate than cells expressing WT IFITM3. Confirming that the amphipathicity of this helix contributes to antiviral activity, single, double and triple alanine mutations of the three residues making up the hydrophilic face of the helix progressively decreased antiviral activity of IFITM3, as did single polar mutations in the hydrophobic face of the helix, or single amino acid insertions that disrupted amphipathicity. Amino acid sequence alignments suggest that amphipathicity in this region is highly conserved in IFITMs throughout evolution. Overall, we utilized bioinformatics methods coupled with functional infectivity assays to provide the first evidence that IFITM3 utilizes an amphipathicity-based mechanism for inhibition of viruses.

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

BackgroundThe ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.ResultsThe 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses.ConclusionThe findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2528-2) contains supplementary material, which is available to authorized users.

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+234Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+234Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.

Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4+ T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels.

We present a web server to predict the functional effect of single or multiple amino acid substitutions, insertions and deletions using the prediction tool PROVEAN. The server provides rapid analysis of protein variants from any organisms, and also supports high-throughput analysis for human and mouse variants at both the genomic and protein levels. The web server is freely available and open to all users with no login requirements at http://provean.jcvi.org. achan@jcvi.org Supplementary data are available at Bioinformatics online.

In Vivo Analysis of Protein Crowding in the Nuclear Pore Complex during Interphase and Mitosis

The nuclear pore complex (NPC) of eukaryotic cells regulates macromolecular traffic between the cytoplasm and the nucleoplasm. The central channel of the pore is thought to form a crowded hydrophobic environment, due to the high content of Phe-Gly (FG) motifs in pore-forming subunits (Nups), which determines the selectivity of the pore. Here, we analyzed the spatiotemporal formation of crowded environments within the NPC by utilizing a crowding-sensitive fluorescent protein probe (GimRET), which was constructed by fusing ECFP with YFP carrying a single amino acid insertion (YFP-G1). The fluorescent properties of GimRET exhibited sensitivity to high concentrations of protein (> ∼100 mg/mL). When GimRET-fused Nups were expressed in HeLa cells. The FRET signal in the nuclear envelop revealed that the extent of crowding is different among Nups. Nups located in the outer rim of the pore (Nups50, 153, 214 and 358) exist in a highly crowded environment, whereas Nups located in the middle channel (Nups54, 58 and 62) exhibited a minimal crowding, suggesting that a large entropic barrier exits at both ends of the pore. We also investigated the formation of a crowded environment during post-mitotic reassembly of the NPC. Quantitative analysis of the probe signals from metaphase to G1 phase indicated that some Nups are in a less crowded environment when dispersed in the cytoplasm at metaphase than when assembled in the NPC during interphase. However, some Nups exhibit a similar amount of crowding in both cases, suggesting that some Nups remain in subcomplexes, but others dissociate during mitosis. In addition, a lag period between the localization of Nups around the chromatin surface and the detection of crowding suggests that there is a dynamic rearrangement of Nups after assembly on the chromatin surface.

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

Hypoxia influences expression profile of Pleckstrin homology-like domain, family A, member 2 in Indian catfish, Clarias batrachus (Linnaeus, 1758): a new candidate gene for hypoxia tolerance in fish.

Several physiologically important genes were found to be regulated by hypoxia at the transcriptional level. The Pleckstrin homology-like domain, family A, member 2 (PHLDA2) gene was previously identified as an imprinted gene. The present study was aimed to determine the structure of complete cDNA and the deduced protein of PHLDA2 along with analysing the changes in its mRNA expression in Clarias batrachus tissues under hypoxic conditions. The complete cDNA of CbPHLDA2 gene consisted of 1009 nucleotides with an open reading frame of 417 nucleotides. The deduced CbPHLDA2 protein of 139 amino acids shared high homology with PHLD2A of other fishes as well as that of vertebrates. Importantly, a single amino acid (asparagine/lysine) insertion was identified in the PH domain of CbPHLDA2 and other fishes, which was absent in other vertebrates studied. Furthermore, under normoxic conditions, CbPHLDA2 was constitutively expressed with varying levels in analysed tissues. Short- and long-term hypoxia exposure resulted in significant changes in the expression of CbPHLDA2 in liver, spleen, head kidney, brain and muscle in a time-dependent manner. The results suggested that CbPHLDA2 might play an important role for adaptive significance under hypoxia.

3P007 Systematic structural study of single amino acid insertion mutants of YFP(01A. Protein: Structure,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

3P007 Systematic structural study of single amino acid insertion mutants of YFP(01A. Protein: Structure,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))