RESUMEN
Bio-semiconductors are expected to be similar to organic semiconductors; however, they have not been utilized in application yet. In this study, we show the origin of electron appearance, N- and S-type negative resistances, rectification, and switching effects of semiconductors with energy storage capacities of up to 418.5 mJ/m2 using granulated amorphous kenaf cellulose particles (AKCPs). The radical electrons in AKCP at 295 K appear in cellulose via the glycosidic bond C1-O1·-C4. Hall effect measurements indicate an n-type semiconductor with a carrier concentration of 9.89 × 1015/cm3, which corresponds to a mobility of 10.66 cm2/Vs and an electric resistivity of 9.80 × 102 Ωcm at 298 K. The conduction mechanism in the kenaf tissue was modelled from AC impedance curves. The light and flexible cellulose-semiconductors may open up new avenues in soft electronics such as switching effect devices and bio-sensors, primarily because they are composed of renewable natural compounds.
RESUMEN
There has been no research conducted thus far on the semiconducting behaviour of biomaterials. In this study, we present an n-type semiconducting biomaterial composed of amorphous kenaf cellulose fibre (AKCF) paper with a voltage-controlled N-type negative resistance. The AKCF generates an alternating-current wave with a frequency of 40.6 MHz from a direct-current voltage source at its threshold voltage (electric field of 5.26 kV/m), which is accompanied by a switching effect with a four-order resistance change at 293 K. This effect is attributed to the voltage-induced occurrence of strong field domains (electric double layers) at the cathode and depletion at the anode of the AKCF device. The proposed AKCF material presents considerable potential for applications in flexible/paper electronic devices such as high frequency power sources and switching effect devices.
Asunto(s)
Suministros de Energía Eléctrica , Electricidad , Electrodos , ElectrónicaRESUMEN
Despite the intense interest in cellulose nanofibers (CNFs) for biomedical and engineering applications, no research findings about the electrical energy storage of CNF have been reported yet. Here, we present the first electroadsorption effects of an amorphous cellulose nanofiber (ACF) supercapacitor, which can store a large amount of electricity (221 mJm-2, 13.1 Wkg-1). The electric storage can be attributed to the entirely enhanced electroadsorption owing to a quantum-size effect by convexity of 17.9 nm, an offset effect caused by positive polar C6=O6 radicles, and an electrostatic effect by appearance of the localised electrons near the Na ions. The supercapacitor also captures both positive and negative electricity from the atmosphere and in vacuum. The supercapacitor could illuminate a red LED for 1 s after charging it with 2 mA at 10 V. Further gains might be attained by integrating CNF specimens with a nano-electromechanical system (NEMS).
RESUMEN
In this study, the electric storage effect of AlO6 clusters in amorphous alumina (AAO) supercapacitors was investigated in terms of cluster morphologies under electron-beam irradiation. Based on first-principles density functional calculation, the optimised structure of AlO6 clusters around an O-vacancy is characterised by a large vacant space created by the absence of an O atom and its neighbouring Al atom. The localised electrons present near the two-atomic vacancies induce positive charges on the inside of the insulating oxide surface, ensuring the adsorption of many electrons on the surface. Electron-beam irradiation (adsorption) from 100 to 180 keV causes the lengths of the Al-O bonds of the cluster to shrink, but then return to the original length with decreasing voltage energy, indicating a rocking-chair-type charge-breathing effect accompanied by a volume expansion of approximately 4%. The I-V and I-R characteristics depicted Coulomb blockade for the switching effect of both the negative and positive potentials. The Ragone plot of the AAO supercapacitor is located at capability area of the second cell.
RESUMEN
Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of â¼â¯5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; ß2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., -0.32â¯ppm for Δδ13Cε [NζD3+-NζH3+] vs. -0.21â¯ppm for Δδ13Cε [NζD2-NζH2] and -1.1â¯ppm for Δδ15Nζ[NζD3+-NζH3+] vs. -1.8â¯ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (>â¯2â¯Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11â¯ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5â¯Hz at a field strength of 14â¯T (150â¯MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.
RESUMEN
Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Mutación Puntual , Agregado de Proteínas , Sitios de Unión/efectos de los fármacos , Glicoles/química , Glicoles/farmacología , Humanos , Proteínas de Complejo Poro Nuclear/metabolismo , Agregado de Proteínas/efectos de los fármacos , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Receptores CCR2/química , Receptores CCR2/metabolismo , SolubilidadRESUMEN
Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.
Asunto(s)
Cadenas Pesadas de Clatrina/metabolismo , Disulfiram/farmacología , Neoplasias Pulmonares/patología , Macrófagos/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Quimiocinas/metabolismo , Progresión de la Enfermedad , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunoterapia , Cinética , Neoplasias Pulmonares/genética , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Metástasis de la Neoplasia , Proteínas de Complejo Poro Nuclear/genética , Pronóstico , Factores de RiesgoRESUMEN
BACKGROUND: The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. METHODS: The 82â¯kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. RESULTS: The MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. CONCLUSIONS: We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. GENERAL SIGNIFICANCE: The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Malato Sintasa/química , Mutación , Proteínas/química , Isótopos de Carbono , Escherichia coli/enzimología , Sustancias Macromoleculares , Péptidos/química , Fenilalanina/química , Estructura Secundaria de Proteína , Protones , Triptófano/químicaRESUMEN
The purpose of this study was to determine the immediate bond effectiveness of universal adhesives to unground and ground enamel surfaces in different etching modes, through shear bond strength (SBS) tests and scanning electron microscopy observations. Three universal adhesives, a conventional two-step self-etch adhesive, and a conventional single-step self-etch adhesive were compared. Human enamel specimens from lower anterior teeth were divided into four groups and subjected to the following treatments: (i) unground enamel in self-etch mode; (ii) ground enamel in self-etch mode; (iii) unground enamel in etch-&-rinse mode; and (iv) ground enamel in etch-&-rinse mode. Bonded assemblies were subjected to SBS testing. All the adhesives showed significantly higher SBS values in etch-&-rinse mode than in self-etch mode, regardless of whether enamel was unground or ground. The influence of the enamel surface condition on SBS was different in different etching modes. Without pre-etching, all tested materials showed lower SBS values in unground enamel than in ground enamel. In etch-&-rinse mode, no significant differences in SBS values were observed between unground enamel and ground enamel for any of the adhesives tested. Phosphoric acid pre-etching before application of self-etch adhesives to an unground enamel surface is essential to enhance initial enamel bond effectiveness.
Asunto(s)
Grabado Ácido Dental , Recubrimiento Dental Adhesivo , Esmalte Dental , Recubrimientos Dentinarios , Adhesivos , Cementos Dentales , Humanos , Ensayo de Materiales , Cementos de Resina , Resistencia al CorteRESUMEN
Water soluble spin-crossover (SCO) iron(II) nanoparticles (NPs) were synthesized by the polyethylene glycol (PEG) coating of [Fe(Htrz)3-3×(NH2trz)3×](BF4)2 (x = 0, 0.1, 0.5 and 1). The NPs with x = 0.1 show gradual SCO behavior over 280-330 K in water. The relaxation times, T1 and T2, were determined and the thermally-responsive T2 values making these NPs a candidate for use as a MRI contrast agent.
RESUMEN
The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Evolución Molecular , Filogenia , Receptores de Superficie Celular/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Receptores de Superficie Celular/metabolismo , Especificidad de la EspecieRESUMEN
In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of "TROSY by isotope labeling" has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52-57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.
Asunto(s)
Isótopos de Carbono , Marcaje Isotópico , Resonancia Magnética Nuclear Biomolecular/métodos , Aminoácidos , Aminoácidos Aromáticos , Conformación ProteicaRESUMEN
FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532-656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain 1H, 13C, and 15N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the 1H-15N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.
Asunto(s)
Quimiotaxis , Citoplasma/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Receptores de Quimiocina/metabolismo , Humanos , Unión ProteicaRESUMEN
The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N- or C-terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants. The solubilizing compounds are then identified from the screened solvents. In this study, the C-terminal chemokine receptor-binding region of the cytoplasmic protein, FROUNT (FNT-C), which mediates intracellular signals leading to leukocyte migration, was subjected to the multicomponent screening. In total, 192 solution conditions were tested, using eight terminal deletion mutants of FNT-C. We identified five solvent conditions that solubilized four or five mutants of FNT-C, and the compounds in the screened solvents were then, respectively, assessed in terms of their solubilization ability. The best compound for solubilizing FNT-C was 1,6-hexanediol. Indeed, 1,6-hexanediol bound to FNT-C and suppressed its precipitation, as showed by NMR and dynamic light scattering analyses.
Asunto(s)
Glicoles/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Estabilidad Proteica , Eliminación de Secuencia , Solventes/metabolismo , Movimiento Celular , Células Cultivadas , Glicoles/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Leucocitos/citología , Leucocitos/fisiología , Mutación , Proteínas de Complejo Poro Nuclear/genética , Multimerización de Proteína/efectos de los fármacos , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Solubilidad , Solventes/químicaRESUMEN
FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.
Asunto(s)
Escherichia coli/metabolismo , Proteínas de Complejo Poro Nuclear/biosíntesis , Proteínas de Complejo Poro Nuclear/química , Receptores CXCR4/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular/métodos , Escherichia coli/genética , Proteínas de Complejo Poro Nuclear/ultraestructura , Unión Proteica , Receptores CXCR4/ultraestructuraRESUMEN
Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14-Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D (13)C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically (13)C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14-Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m c14 and m c38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for (13)C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the (1)H-(13)C signals at the ß-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.
Asunto(s)
Aprotinina/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Disulfuros/química , Resonancia Magnética Nuclear Biomolecular , Algoritmos , Animales , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Bovinos , Isoenzimas , Modelos Químicos , Estructura Molecular , Proteínas Mutantes , Resonancia Magnética Nuclear Biomolecular/métodos , Temperatura , TermodinámicaRESUMEN
We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and ß-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,ß-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,ß-dihydroxy-α-methylvalerate to α-keto-ß-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.
Asunto(s)
Escherichia coli , Isoleucina/química , Marcaje Isotópico , Leucina/química , Espectroscopía de Resonancia Magnética , Proteínas/química , Valina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Isoleucina/metabolismo , Leucina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/metabolismo , Valina/metabolismoRESUMEN
The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.
Asunto(s)
Inmunosupresores/metabolismo , Sirolimus/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Tacrolimus/metabolismo , Humanos , Enlace de Hidrógeno , Inmunosupresores/química , Ligandos , Modelos Moleculares , Movimiento (Física) , Unión Proteica , Conformación Proteica , Sirolimus/química , Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/química , TermodinámicaRESUMEN
We present a 3D (1)H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10-100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at â¼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers â¼110 fold time saving over a traditional 3D (13)C-detected SSNMR approach.