RESUMEN
Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.
Asunto(s)
Calcio , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismo , Músculo Esquelético/metabolismo , Canales de Calcio Tipo L/análisis , Canales de Calcio Tipo L/metabolismo , Retículo Sarcoplasmático/metabolismo , Contracción Muscular/fisiologíaRESUMEN
Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increased successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation as a layer of holey supporting film covering on TEM grid. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including much better electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.
RESUMEN
Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increased successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation as a layer of holey supporting film covering on TEM grid. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including much better electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.
Asunto(s)
Aleaciones/química , Microscopía por Crioelectrón/métodos , Níquel/química , Titanio/química , Animales , Apoferritinas/química , Antígeno B7-H1/química , Materiales Biocompatibles , Biotinilación , Carbono , Oro , Hipocampo/metabolismo , Humanos , Ligandos , Campos Magnéticos , Ensayo de Materiales , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Movimiento (Física) , Neuronas/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Silicio/química , Estreptavidina/química , TemperaturaRESUMEN
Chlorantraniliprobe (Chlo), a potent insecticide, demolishes intracellular Ca2+ homeostasis of insects by inducing uncontrolled Ca2+ release through ryanodine receptors (RyRs). Chlo is lethal to insects but has low toxicity to mammals. In this study, we investigated the effects of Chlo on RyR1 from mammalian skeletal muscle. Ca2+ release assay indicated that Chlo at high concentrations promoted Ca2+ release from sarcoplasmic reticulum through RyR1 channels. Single channel recording of purified RyR1 showed that Chlo activated RyR1 channel, increased channel open probability Po, reduced channel mean close time Tc, but did not change the channel mean open time To, suggesting that Chlo destabilized the closed RyR1 channel, rendered the channel easy to open. The dissociation constant Kd values of Chlo for RyR1 were of micromolar level, approximately 100-fold larger than that for insect RyR. The Kd values were smaller for open states than for closed/blocked states of the RyR1 channel. The maximal binding capacity Bmax did not change in the presence of either channel activators or inhibitors/blockers. Our results demonstrate that the insecticide Chlo is a weak activator of mammalian RyR1. It can interact with mammalian RyR1 and activate RyR1 channel but with much lower affinity compared with insect RyR; Chlo has a binding site distinct from all known RyR channel modulators and represents a novel type of RyR channel modulator. Our data provide biochemical and pharmacological insights into its high specificity to insect RyR and high selectivity of poisoning to insects over mammals.
Asunto(s)
Insecticidas/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ortoaminobenzoatos/farmacología , Animales , Calcio/metabolismo , ConejosRESUMEN
Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETaseS93M, IsPETaseW159F, and IsPETaseN241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/ß-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.
Asunto(s)
Burkholderiales/enzimología , Hidrolasas/metabolismo , Tereftalatos Polietilenos/metabolismo , Hidrolasas/química , Hidrolasas/genética , Modelos Moleculares , Tereftalatos Polietilenos/química , Conformación ProteicaRESUMEN
Hepatitis B Virus core protein (HBc) has multiple roles in the viral lifecycle: viral assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions. HBc displays assembly polymorphism - it can assemble into icosahedral capsid and aberrant non-capsid structures. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit, however, the mechanism governing the polymorphic assembly of the HBc dimer is still elusive. By using the experimental antiviral drug BAY 41-4109, we successfully transformed the HBc assembly from icosahedral capsid to helical tube. Structural analyses of HBc dimers from helical tubes, T = 4 icosahedral capsid, and sheet-like HBc ensemble revealed differences within the inter-dimer interface. Disruption of the HBc inter-dimer interface may likely promote the various assembly forms of HBc. Our work provides new structural insights into the HBV assembly mechanism and strategic guide for anti-HBV drug design.
Asunto(s)
Virus de la Hepatitis B/química , Virus de la Hepatitis B/fisiología , Proteínas del Núcleo Viral/química , Ensamble de Virus , Escherichia coli , Humanos , Modelos Moleculares , Multimerización de Proteína , Estructura Terciaria de Proteína , Piridinas/farmacología , Pirimidinas/farmacología , Proteínas del Núcleo Viral/ultraestructuraRESUMEN
In single-particle cryo-electron microscopy (cryo-EM), K-means clustering algorithm is widely used in unsupervised 2D classification of projection images of biological macromolecules. 3D ab initio reconstruction requires accurate unsupervised classification in order to separate molecular projections of distinct orientations. Due to background noise in single-particle images and uncertainty of molecular orientations, traditional K-means clustering algorithm may classify images into wrong classes and produce classes with a large variation in membership. Overcoming these limitations requires further development on clustering algorithms for cryo-EM data analysis. We propose a novel unsupervised data clustering method building upon the traditional K-means algorithm. By introducing an adaptive constraint term in the objective function, our algorithm not only avoids a large variation in class sizes but also produces more accurate data clustering. Applications of this approach to both simulated and experimental cryo-EM data demonstrate that our algorithm is a significantly improved alterative to the traditional K-means algorithm in single-particle cryo-EM analysis.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Análisis por Conglomerados , Microscopía por Crioelectrón/normas , Relación Señal-RuidoRESUMEN
Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.
Asunto(s)
Calcio/farmacología , Activación del Canal Iónico , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Motivos EF Hand , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Dominios Proteicos , Conejos , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Relación Estructura-ActividadRESUMEN
Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/química , Ribosomas/química , Simulación por Computador , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The matrix protein 1 (M1) is the most abundant structural protein in influenza A virus particles. It oligomerizes to form the matrix layer under the lipid membrane, sustaining stabilization of the morphology of the virion. The present study indicates that M1 forms oligomers based on a fourfold symmetrical oligomerization pattern. Further analysis revealed that the oligomerization pattern of M1 was controlled by a highly conserved region within the C-terminal domain. Two polar residues of this region, serine-183 (S183) and threonine-185 (T185), were identified to be critical for the oligomerization pattern of M1. M1 point mutants suggest that single S183A or T185A substitution could result in the production of morphologically filamentous particles, while double substitutions, M1-S183A/T185A, totally disrupted the fourfold symmetry and resulted in the failure of virus production. These data indicate that the polar groups in these residues are essential to control the oligomerization pattern of M1. Thus, the present study will aid in determining the mechanisms of influenza A virus matrix layer formation during virus morphogenesis.
Asunto(s)
Virus de la Influenza A/fisiología , Proteínas de la Matriz Viral/metabolismo , Virión/metabolismo , Ensamble de Virus , Aminoácidos/genética , Animales , Línea Celular , Análisis Mutacional de ADN , Perros , Humanos , Virus de la Influenza A/genética , Mutación Puntual , Multimerización de Proteína , Proteínas de la Matriz Viral/genéticaRESUMEN
Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and single particle 3D reconstruction that, at the low temperatures (4-25°C), CeHSP17 (a sHSP from Caenorhabditis elegans) exists as a 24-subunit spherical oligomer with tetrahedral symmetry. Our studies demonstrate that CeHSP17 forms large sheet-like super-molecular assemblies (SMAs) at the high temperatures (45-60°C), and such SMAs are apparently the form that exhibits chaperone-like activity. Our findings suggest a novel molecular mechanism for sHSPs to function as molecular chaperones.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/química , Chaperonas Moleculares/genética , Adaptación Biológica , Animales , Caenorhabditis elegans , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Proteínas Recombinantes , TemperaturaRESUMEN
To obtain a comprehensive understanding of proteins involved in mitochondrion-sarcoplasmic reticulum (SR) linking, a catalog of proteins from mitochondrion-associated membrane (MAM) of New Zealand white rabbit skeletal muscle were analyzed by an optimized shotgun proteomic method. The membrane fractions were prepared by differential centrifugation and separated by 1D electrophoresis followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ)-Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 459 proteins were identified from both of the two independent MAM preparations. Protein pI value, molecular weight range, and transmembrane region were calculated using bioinformatics softwares. One hundred one proteins were recognized as membrane proteins. This protein database suggested that the MAM preparations composed of proteins from mitochondrion, SR, and transverse-tubule. This result indicated mitochondria physically linked with SR in rabbit skeletal muscle, voltage-dependent anion channel 1 (VDAC1), VDAC2, and VDAC3 might participate in formation of the tethers between SR and mitochondria.
Asunto(s)
Mitocondrias Musculares/metabolismo , Membranas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Retículo Sarcoplasmático/metabolismo , Animales , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Espectrometría de Masas en Tándem , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Dihydropyridine receptor (DHPR), an L-type Ca(2+) channel complex, plays an essential role in muscle contraction, secretion, integration of synaptic input in neurons and synaptic transmission. The molecular architecture of DHPR complex remains elusive. Here we present a 15-Å resolution cryo-electron microscopy structure of the skeletal DHPR/L-type Ca(2+) channel complex. The DHPR has an asymmetrical main body joined by a hook-like extension. The main body is composed of a "trapezoid" and a "tetrahedroid". Homologous crystal structure docking and site-specific antibody labelling revealed that the α1 and α2 subunits are located in the "trapezoid" and the ß subunit is located in the "tetrahedroid". This structure revealed the molecular architecture of a eukaryotic Ca(2+) channel complex. Furthermore, this structure provides structural insights into the key elements of DHPR involved in physical coupling with the RyR/Ca(2+) release channel and shed light onto the mechanism of excitation-contraction coupling.
Asunto(s)
Canales de Calcio Tipo L/química , Membrana Celular/química , Proteínas Musculares/química , Músculo Esquelético/química , Canal Liberador de Calcio Receptor de Rianodina/química , Animales , Canales de Calcio Tipo L/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Microscopía por Crioelectrón , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Proteínas de Choque Térmico/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular , Escherichia coli/genética , Eliminación de Gen , Proteínas de Choque Térmico/genética , Calor , Viabilidad Microbiana , Mutación , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: This study was designed to investigate the protective potential of AS-IV against ischemia and I/R-induced myocardial damage, with focusing on possible involvement of energy metabolism modulation in its action and the time phase in which it takes effect. METHODS: SD rats were subjected to 30 minutes LADCA occlusion, followed by reperfusion. MBF, myocardial infarct size, and cardiac function were evaluated. Myocardial structure and myocardial apoptosis were assessed by double immunofluorescence staining of F-actin and TUNEL. Content of ATP, ADP, and AMP in myocardium, cTnI level, expression of ATP5D, P-MLC2, and apoptosis-related molecules were determined. RESULTS: Pretreatment with AS-IV suppressed MBF decrease, myocardial cell apoptosis, and myocardial infarction induced by I/R. Moreover, ischemia and I/R both caused cardiac malfunction, decrease in the ratio of ATP/ADP and ATP/AMP, accompanying with reduction of ATP 5D protein and mRNA, and increase in P-MLC2 and serum cTnI, all of which were significantly alleviated by pretreatment with AS-IV, even early in ischemia phase for the insults that were implicated in energy metabolism. CONCLUSIONS: AS-IV prevents I/R-induced cardiac malfunction, maintains the integrity of myocardial structure through regulating energy metabolism. The beneficial effect of AS-IV on energy metabolism initiates during the phase of ischemia.
Asunto(s)
Daño por Reperfusión Miocárdica , Miocardio , Saponinas/farmacología , Triterpenos/farmacología , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Masculino , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Sprague-Dawley , Troponina I/biosíntesisRESUMEN
Cryo-electron microscopy (cryo-EM) now plays an important role in structural analysis of macromolecular complexes, organelles and cells. However, the cryo-EM images obtained close to focus and under low dose conditions have a very high level of noise and a very low contrast, which hinders high-resolution structural analysis. Here, an optimized locally adaptive non-local (LANL) means filter, which can preserve signal details and simultaneously significantly suppress noise for cryo-EM data, is presented. This filter takes advantage of a wide range of pixels to estimate the denoised pixel values instead of the traditional filter that only uses pixels in the local neighborhood. The filter performed well on simulated data and showed promising results on raw cryo-EM images and tomograms. The predominant advantage of this optimized LANL-means filter is the structural signal and the background are clearly distinguishable. This locally adaptive non-local means filter may become a useful tool in the analysis of cryo-EM data, such as automatic particle picking, extracting structural features and segmentation of tomograms.
Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodosRESUMEN
Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.
Asunto(s)
Canales de Cloruro/metabolismo , Canales de Cloruro/ultraestructura , Microscopía por Crioelectrón , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Animales , Calcio/metabolismo , Cristalografía por Rayos X , Activación del Canal Iónico , Modelos Moleculares , Músculo Esquelético/metabolismo , Unión Proteica , Conformación Proteica , Conejos , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/aislamiento & purificación , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructura , Propiedades de Superficie , TritioRESUMEN
The randomization scheme of hypervariable region takes crucial role in construction of a synthetic antibody library. The codon bias and inevitable 'stop' codon of conventional 'NNK' and 'NNS' codons limit their applications. Here we report a split-mix-split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56 x 10(9) was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that >60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 x anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split-mix-split DNA randomization strategy in library construction and site-directed mutagenesis.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Región Variable de Inmunoglobulina/genética , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Anticuerpos , Regiones Determinantes de Complementariedad/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The authors investigate the effects and mechanisms of the anti-hepatitis B virus (HBV) agent Bay 41-4109. HepG2.2.15 cells were used to investigate the antiviral effects of Bay 41-4109 by using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence. The C terminally truncated core protein was expressed and purified. Changes in hepatitis B capsid formation were assayed by dynamic light scattering and transmission electronic microscopy. Bay 41-4109 was found to be a highly selective and potent inhibitor of hepatitis B virus replication in HepG2.2.15 cells. This compound was equally effective at inhibiting HBV DNA release and the cytoplasmic HBcAg level, with 50% inhibitory concentrations of 32.6 and 132 nM, respectively. HBV DNA and HBcAg were inhibited in a dose-dependent manner, indicating that the anti-HBV mechanisms are associated with and dependent on the rate of HBcAg inhibition. Our results indicate that Bay 41-4109 treatment disassembled the core capsids and separated them into monomers or dimers, the form in which they could be further degraded into peptides. The core protein assembled in a misdirected manner cannot function effectively. Our results suggest that, based on its particular activities, Bay 41-4109 is a promising anti-HBV candidate.