ABSTRACT
Dabrafenib (DBF), an anticancer drug, exhibits isostructural properties in its hydrate (DBFâH2O) and perhydrate (DBFâH2O2) forms, as revealed by single-crystal X-ray diffraction. Despite the H2O and H2O2 solvent molecules occupying identical locations, the two polymorphs have different thermal behaviors. In general, determination of stoichiometry of H2O in the perhydrate crystals is difficult due to the presence of both H2O and H2O2 in the same crystal voids. This study utilizes magic-angle spinning (MAS) solid-state NMR (SSNMR) combined with gauge-included projector augmented wave calculations to characterize the influence of solvent molecules on the local environment in pseudopolymorphs. SSNMR experiments were employed to assign 1H, 13C, and 15N peaks and identify spectral differences in the isostructural pseudopolymorphs. Proton spectroscopy at fast MAS was used to identify and quantify H2O2/H2O in DBFâH2O2 (mixed hydrate/perhydrate). 1H-1H dipolar-coupling-based experiments were recruited to confirm the 3D molecular packing of solvent molecules in DBFâH2O and DBFâH2O2. Homonuclear (1H-1H) and heteronuclear (1H-14N) distance measurements, in conjunction with diffraction structures and optimized hydrogen atom positions by density functional theory, helped decipher local interactions of H2O2 with DBF and their geometry in DBFâH2O2. This integrated X-ray structure, quantum chemical calculations, and NMR study of pseudopolymorphs offer a practical approach to scrutinizing crystallized solvent interactions in the crystal lattice even without high-resolution crystal structures or artificial sample enrichment.
ABSTRACT
Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.
Subject(s)
Proton Pumps , Rhodopsin , Rhodopsin/chemistry , Proton Pumps/chemistry , Hydrogen Bonding , Magnetic Resonance SpectroscopyABSTRACT
Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR 'fingerprints' as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31-119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs.
Subject(s)
Nucleic Acid Conformation , RNA, Long Noncoding/ultrastructure , Retroelements/genetics , Short Interspersed Nucleotide Elements/genetics , Computational Biology , Humans , Magnetic Resonance Spectroscopy , RNA, Antisense/genetics , RNA, Antisense/ultrastructure , RNA, Long Noncoding/genetics , Transcriptome/genetics , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/ultrastructureABSTRACT
NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Membrane Proteins , Models, Molecular , Protein Conformation , Software , Computational Biology , Machine Learning , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Folding , Sequence Analysis, ProteinABSTRACT
Amyloid-ß (Aß) fibrils in neuritic plaques are a hallmark of Alzheimer's disease (AD). Since the 42-residue Aß (Aß42) fibril is the most pathogenic among different Aß species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aß40, previous studies of Aß42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped ß-sheet arrangement (e.g., Xiao et al. Nat. Struct. Mol. Biol. 2015, 22, 499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aß42 fibril prepared with â¼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aß42 (â¼40 nmol or â¼200 µg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from â¼1 pmol of brain amyloid seed in â¼2.5 h. We also present the first structural characterization of synthetic fully protonated Aß42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (â¼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aß42 fibril exhibits a novel fold or polymorph structure.
Subject(s)
Amyloid beta-Peptides/chemistry , Nuclear Magnetic Resonance, Biomolecular , Amyloid beta-Peptides/metabolism , Brain/metabolism , Humans , Protein Conformation , ProteinsABSTRACT
Three-dimensional electron diffraction crystallography (microED) can solve structures of sub-micrometer crystals, which are too small for single crystal X-ray crystallography. However, R factors for the microED-based structures are generally high because of dynamic scattering. That means R factor may not be reliable provided that kinetic analysis is used. Consequently, there remains ambiguity to locate hydrogens and to assign nuclei with close atomic numbers, like carbon, nitrogen, and oxygen. Herein, we employed microED and ssNMR dipolar-based experiments together with spin dynamics numerical simulations. The NMR dipolar-based experiments were 1H-14N phase-modulated rotational-echo saturation-pulse double-resonance (PM-S-RESPDOR) and 1H-1H selective recoupling of proton (SERP) experiments. The former examined the dephasing effect of a specific 1H resonance under multiple 1H-14N dipolar couplings. The latter examined the selective polarization transfer between a 1H-1H pair. The structure was solved by microED and then validated by evaluating the agreement between experimental and calculated dipolar-based NMR results. As the measurements were performed on 1H and 14N, the method can be employed for natural abundance samples. Furthermore, the whole validation procedure was conducted at 293 K unlike widely used chemical shift calculation at 0 K using the GIPAW method. This combined method was demonstrated on monoclinic l-histidine.
ABSTRACT
Aquaporins are integral membrane proteins that facilitate water flow across biological membranes. Their involvement in multiple physiological functions and disease states has prompted intense research to discover water channel activity modulators. However, inhibitors found so far are weak and/or lack specificity. For organic compounds, which lack of high electron-dense atoms, the identification of binding sites is even more difficult. Nuclear magnetic resonance spectroscopy (NMR) requires large amounts of the protein, and expression and purification of mammalian aquaporins in large quantities is a difficult task. However, since aquaporin Z (AqpZ) can be purified and expressed in good quantities and has a high similarity to human AQP1 (~ 40% identity), it can be used as a model for studying the structure and function of human aquaporins. In the present study, we have used solid-state MAS NMR to investigate the binding of a lead compound [1-(4-methylphenyl)1H-pyrrole-2,5-dione] to AqpZ, through mapping of chemical shift perturbations in the presence of the compound.
Subject(s)
Aquaporins/antagonists & inhibitors , Nuclear Magnetic Resonance, Biomolecular/methods , Animals , Aquaporin 1/chemistry , Aquaporin 1/metabolism , Humans , Mammals , Protein Binding , Pyrroles/metabolism , Pyrroles/pharmacologyABSTRACT
Mycobacteriophage archival stocks have been kept for ca. 20-50 years in Japan. In this study, we attempted to recover mycobacteriophages from 50 archival stocks and briefly analyzed the recovered phages. The phages were recovered from 72.2% (13/18) of the lyophilized stocks that had been stored for 47-56 years. Moreover, the analysis of 12 representative recovered phages led to their classification as belonging to the family Siphoviridae, and seven of them were typed by polymerase chain reaction (PCR) targeting the gene that encodes the tape measure protein. Considering these results, lyophilization seems to be suitable for phage archival storage.
Subject(s)
Biological Specimen Banks , Mycobacteriophages/classification , Mycobacteriophages/isolation & purification , Bacteriological Techniques , Freeze Drying , Genome, Viral , Japan , Mycobacteriophages/genetics , Mycobacteriophages/ultrastructure , Mycobacterium smegmatis/virology , Polymerase Chain Reaction , Siphoviridae/classification , Siphoviridae/genetics , Siphoviridae/isolation & purification , Siphoviridae/ultrastructure , Specimen Handling/methods , Viral Proteins/geneticsABSTRACT
Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor ß-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor ß-induced protein (amino acid 611-642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.
Subject(s)
Cornea/metabolism , Corneal Dystrophies, Hereditary/metabolism , Eye Proteins/metabolism , Peptide Fragments/metabolism , Point Mutation , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta/metabolism , Up-Regulation , Amino Acid Substitution , Amyloid/chemistry , Amyloid/genetics , Amyloid/metabolism , Amyloid/ultrastructure , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/genetics , Amyloidogenic Proteins/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid , Circular Dichroism , Cornea/cytology , Cornea/pathology , Cornea/ultrastructure , Corneal Dystrophies, Hereditary/genetics , Corneal Dystrophies, Hereditary/pathology , Eye Proteins/chemistry , Eye Proteins/genetics , Humans , Kinetics , Microscopy, Electron, Transmission , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/metabolism , Protein Interaction Domains and Motifs , Protein Stability , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Transforming Growth Factor beta/chemistry , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1/chemistry , Transforming Growth Factor beta1/geneticsABSTRACT
Eukaryotic chromatin structure and dynamics play key roles in genomic regulation. In the current study, the secondary structure and intramolecular dynamics of human histone H4 (hH4) in the nucleosome core particle (NCP) and in a nucleosome array are determined by solid-state NMR (SSNMR). Secondary structure elements are successfully localized in the hH4 in the NCP precipitated with Mg2+ . In particular, dynamics on nanosecond to microsecond and microsecond to millisecond timescales are elucidated, revealing diverse internal motions in the hH4 protein. Relatively higher flexibility is observed for residues participating in the regulation of chromatin mobility and DNA accessibility. Furthermore, our study reveals that hH4 in the nucleosome array adopts the same structure and show similar internal dynamics as that in the NCP assembly while exhibiting relatively restricted motions in several regions consisting of residues in the N-terminus, Loop 1, and the α3 helix region.
Subject(s)
Nucleosomes , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/metabolismABSTRACT
Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.
Subject(s)
Antibodies/blood , Antibodies/chemistry , Magnetic Resonance Spectroscopy , Antibodies/immunology , Antibody Specificity , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Isotope Labeling , Models, Molecular , Protein ConformationABSTRACT
BACKGROUND: Levocarnitine deficiency in hemodialysis patients is common. Although the effect of levocarnitine therapy on uremic anemia has been studied in small trials, its effects on cardiac function remain unclear. STUDY DESIGN: Multicenter, prospective, open-label, parallel, randomized, controlled trial. SETTING & PARTICIPANTS: Patients undergoing maintenance hemodialysis with carnitine deficiency (free carnitine plasma concentration < 40µmol/L) enrolled in 3 hemodialysis centers. INTERVENTION: Random assignment to treatment for 12 months with oral levocarnitine therapy at a dose of 20mg/kg/d or control group (no levocarnitine therapy). OUTCOMES & MEASUREMENTS: Cardiac function was assessed by echocardiography. The primary end point was change in ejection fraction from baseline at the end of the study. Secondary end points included changes in left ventricular mass index and clinical parameters from baseline at the end of the study. RESULTS: 222 patients were randomly assigned, of whom 148 patients (levocarnitine group, n=75; control group, n=73) were analyzed. Ejection fraction increased from baseline to the end of the study in the levocarnitine group by 5.43% (95% CI, 4.53%-6.32%), but not in the control group (change, -0.14%; between-group difference, 5.57% [95% CI, 4.48%-6.66%]; P<0.001). Left ventricular mass index decreased from baseline to the end of the study in the levocarnitine group (change of -8.89 [95% CI, -11.7 to -6.09] g/m(2)), but not in the control group (change of 1.62g/m(2); between-group difference, 10.50 [95% CI, 7.51 to 13.60] g/m(2); P<0.001). Levocarnitine therapy reduced N-terminal pro-brain natriuretic peptide (NT-proBNP) levels and improved the erythropoietin responsiveness index, whereas no such effects were observed in the control group. LIMITATIONS: Not a double-blinded study. CONCLUSIONS: Levocarnitine therapy is useful for hemodialysis patients with carnitine deficiency; these patients may benefit from such therapy, with amelioration of cardiac function and reduction of left ventricular mass index.
Subject(s)
Carnitine/therapeutic use , Hypertrophy, Left Ventricular/blood , Hypertrophy, Left Ventricular/therapy , Kidney Failure, Chronic/blood , Kidney Failure, Chronic/therapy , Renal Dialysis/trends , Aged , Aged, 80 and over , Carnitine/blood , Carnitine/deficiency , Female , Humans , Hypertrophy, Left Ventricular/diagnosis , Kidney Failure, Chronic/diagnosis , Male , Middle Aged , Prospective Studies , Renal Dialysis/methodsABSTRACT
Control over macromolecular structure offers bright potentials for manipulation of macromolecular functions. We here present structure-correlation NMR spectroscopy to analyze the correlation between polymorphic macromolecular structures driven by photoisomerization of azobenzene. The structural conversion of azobenzene was induced within the mixing time of a NOESY experiment using a colored light source, and the reverse structural conversion was induced during the relaxation delay using a light source of another color. The correlation spectrum between trans- and cis-azobenzene was then obtained. To maximize the efficiency of the bidirectional photoisomerization of azobenzene-containing macromolecules, we developed a novel light-irradiation NMR sample tube and method for irradiating target molecules in an NMR radio frequency (rf) coil. When this sample tube was used for photoisomerization of an azobenzene derivative at a concentration of 0.2 mM, data collection with reasonable sensitivity applicable to macromolecules was achieved. We performed isomerization of an azobenzene-cross-linked peptide within the mixing time of a NOESY experiment that produced cross-peaks between helix and random-coil forms of the peptide. Thus, these results indicate that macromolecular structure manipulation can be incorporated into an NMR pulse sequence using an azobenzene derivative and irradiation with light of two types of wavelengths, providing a new method for structural analysis of metastable states of macromolecules.
Subject(s)
Azo Compounds/analysis , Azo Compounds/chemistry , Macromolecular Substances/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Photochemical Processes , Reference Standards , StereoisomerismABSTRACT
UNLABELLED: This study aimed to determine the population pharmacokinetics of doxapram in low-birth-weight (LBW) infants. A total of 92 serum concentration measurements that were obtained from 34 Japanese neonates were analyzed using nonlinear mixed-effect modeling (NONMEM). Estimates generated by NONMEM indicated that clearance of doxapram (CL; L/kg/h) was affected by postmenstrual age (PMA; weeks), body weight (BW; g), and aspartate aminotransferase (AST; IU/L). In addition, the volume of distribution (Vd; L/kg) was affected by gestational age (GA; weeks). The final pharmacokinetic model was as follows: CL = BW / PMA × 0.0453 × serum AST(-0.373); Vd = 2.54 (if GA >28 weeks) and Vd = 2.54 × 2.11 (if GA ≤28 weeks). The interindividual variabilities in CL and Vd were 39.9 and 83.0 %, respectively, and the residual variability was 20.9 %. To clarify the reasons for large interindividual variations, the enzymes involved in the metabolic pathway of doxapram were also determined. We found that doxapram was metabolized by CYP3A4/5. CONCLUSION: We report the population pharmacokinetics of doxapram in neonates and the involvement of CYP3A4/5 in its metabolism. The final model of population pharmacokinetics may be useful for formulating a safe and effective dosage regimen and for predicting serum doxapram concentrations in neonates.
Subject(s)
Apnea/metabolism , Central Nervous System Stimulants/pharmacokinetics , Doxapram/pharmacokinetics , Infant, Low Birth Weight , Apnea/drug therapy , Asian People , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System , Double-Blind Method , Female , Humans , Infant, Newborn , Infant, Premature , Japan , Male , Mass Spectrometry , Models, BiologicalABSTRACT
Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between ß1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.
Subject(s)
Amino Acids, Aromatic/chemistry , Nerve Tissue Proteins/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Animals , Base Sequence , Binding Sites , Consensus Sequence , Mice , Models, Molecular , Nerve Tissue Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Four years has passed since QuantiFERON TB-Gold In-Tube (QFT-GIT), the third generation test, has replaced QuantiFERON-Gold in Japan. The QFT-GIT test detects interferon-gamma (IFN-γ), which is released from lymphocytes present in blood after exposure to the M. tuberculosis complex antigens ESAT-6, CFP-10 and TB7.7. These proteins are absent from all Bacille-Calmette-Guérin (BCG) strains and from most non-tuberculosis mycobacteria, resulting in fewer false positive reactions as seen with the tuberculin skin test (TST). We had various experiences with QFT-GIT during these four years. So, we discussed the usefulness and its limitation of QFT-GIT as follows: 1. Development of the principle of QuantiFERON-GIT: Nobuyuki HARADA (Research Institute of Immune Diagnosis (RIID)). QuantiFERON (QFT) was originated from diagnostic system for bovine in Australia. Although the first generation of QFT, in which PPD had been used as stimulating antigens, was approved in USA, its diagnostic value was not recognized in Japan where most of Japanese are vaccinated with BCG. By combining M. tuberculosis-specific antigens with QFT system, the second generation of QFT, QFT-Gold, was developed, and approved in Japan in 2005. QFT-Gold was soon incorporated in several guidelines such as contact investigations and nosocomial infection measures. Now, QFT-Gold was superseded by the improved QFT-Gold, the current QFT-GIT. However, since QFT-GIT may contain unstable factors including blood volume and shaking methods of blood collection tubes, development of the more improved version is strongly expected. 2. Evaluating the result of QFT-GIT in patients treated with dialysis and immunosuppressive agents: Hidetoshi IGARI (National Hospital Organization Chiba-East National Hospital) The effectiveness of QuantiFERON TB-Gold In-Tube was analyzed in the patients with chronic kidney disease (CKD) and rheumatoid arthritis (RA). QFT positive was 7% and 11% respectively, and indeterminate was 5% and 2% respectively. QFT positive was 2% in hemodialysis patients, significantly lower than that of CKD. QFT positive after biological drug was administered was 8% in RA patients, significantly lower than 15% of RA without biological drug. The rate of latent tuberculosis patients in CKD was as well as health care workers (HCWs) of 8% of QFT positive. On the other hand that of RA might be higher than HCWs. Hemodialysis and biological drug administration might attenuate QFT result with lower rate of positive. The rate of indeterminate was less than 5%. This results was improved in compared with former generation QFT. 3. QFT in Vietnam: Naoto KEICHO (Research Institute of Tuberculosis, JATA). We have promoted collaborative research on tuberculosis with Vietnamese institutes since 2002. NCGM-BMH Medical Collaboration Center plays an important role in the clinical research projects. We report 1) quality assessment of QFT for tuberculosis infection, 2) prevalence and risk factors for tuberculosis infection among hospital workers, and 3) analysis of factors lowering sensitivity of QFT for active tuberculosis. We also discuss significance of QFT in developing countries. 4. Comparison of diagnostic performances using QFT Gold and Gold In-Tube in patients with active tuberculosis: Tetsuya YAGI (Department of Infectious Diseases, Center of National University Hospital for Infection Control, Nagoya University Hospital). The goal of this study was to assess the diagnostic performances of QFT-GIT compared with QFT-Gold in patients with active tuberculosis in Nagoya University Hospital, in Japan. The sensitivity of QFT-Gold was 87.2%, the specificity of that was 77.5%. The sensitivity of QFT-GIT was 88.8%, specificity 73.2%. The performance of QFT-GIT was the same as that of QFT-Gold. The QFT-GIT tended to show higher concentration values of IFN-γ than that of QFT-Gold especially in patients with extra pulmonary tuberculosis, smear positive pulmonary tuberculosis, both lung lesion and using immunosuppressive medications. 5. Simultaneous and longitudinal comparison between QFT Gold and Gold In-Tube among health care workers; Tomoshige MATSUMOTO (Department of Clinical Laboratory Medicine, Osaka Anti-Tuberculosis Association Osaka Hospital. ex-Osaka Prefectural Medical Center for Respiratory and Allergic Diseases). The aim of this study was to compare the indeterminate rates between QFT-GIT and QFT-Gold tests. And to make longitudinal comparison by QFT-Gold assay to the same HCW. We collected blood samples by simultaneously QFT-Gold and QFT-GIT from 120 staff members in the institute who participated in this prospective comparison study. Moreover, the latest QFT-Gold test was longitudinally compared for the same 55 staff members who have received QFT-Gold before. The statistically significant difference was observed in the results of indeterminate rate between QFT-Gold and QFT-GIT using the same blood samples. It is concluded that QFT-Gold and QFT-GIT are different assays therefore it is difficult to compare QFT-Gold with QFT-GIT data on the same level. Concerning the follow-up test of the 55 people by QFT-Gold, 5 turned from positive to negative and 4 turned from indeterminate to negative. From this analysis, QFT-Gold positive subjects in the previous time have not been always positive. 6. Interpreting QFT "equivocal" results: Kenji MATSUMOTO (Osaka City Public Health Office). The participants were examined QFT-GIT test after two months to four months from last contact of smear-positive tuberculosis cases in contact investigations. We enrolled 79 contacts whose tests of QFT-GIT were equivocal results. The second QFT-GIT results were 42 negative (53.2%), 28 equivocal (35.4%) and nine positive (11.4%). 64% of the second QFT-GIT tests result in negative or positive among the first QFT-GIT equivocal contacts. When the second QFT-GIT tests were positive, it is highly probable that the contacts were infected tuberculosis and we adequately could treat latent tuberculosis infected contacts.
Subject(s)
Tuberculin Test/methods , Tuberculosis/diagnosis , HumansABSTRACT
Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.
Subject(s)
Bacterial Proteins , Protein Multimerization , Serine-Type D-Ala-D-Ala Carboxypeptidase , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/antagonists & inhibitors , Catalytic Domain , Metalloendopeptidases/chemistry , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Serine-Type D-Ala-D-Ala Carboxypeptidase/chemistry , Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/physiology , Vancomycin Resistance/genetics , Methicillin-Resistant Staphylococcus aureus/enzymology , Methicillin-Resistant Staphylococcus aureus/metabolismABSTRACT
Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by 15N-1H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. These NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.
ABSTRACT
Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.
Subject(s)
Retinaldehyde , Thermoplasmales , Retinaldehyde/chemistry , Retinaldehyde/metabolism , Schiff Bases/chemistry , Rhodopsin/chemistry , Rhodopsin/metabolism , Rhodopsins, Microbial/chemistry , Magnetic Resonance Spectroscopy/methods , Thermoplasmales/metabolism , Archaea/metabolismABSTRACT
To investigate potential applications of the 3,3'-dihydroxy-2,2'-biindan-1,1'-dione (BIT) structure as an organic semiconductor with intramolecular hydrogen bonds, a new synthetic route under mild conditions is developed based on the addition reaction of 1,3-dione to ninhydrin and the subsequent hydrogenation of the hydroxyl group. This route affords several new BIT derivatives, including asymmetrically substituted structures that are difficult to access by conventional high-temperature synthesis. The BIT derivatives exhibit rapid tautomerization by intramolecular double proton transfer in solution. The tautomerizations are also observed in the solid state by variable temperature measurements of X-ray diffractometry and magic angle spinning 13C solid-state NMR. Possible interplay between the double proton transfer and the charge transport is suggested by quantum chemical calculations. The monoalkylated BIT derivative with a lamellar packing structure suitable for lateral charge transport in films shows a hole mobility of up to 0.012 cm2 V-1 s-1 with a weak temperature dependence in an organic field effect transistor.