RESUMEN
Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.
Asunto(s)
Calcio , Calmodulina , Nanoporos , Humanos , Calcio/análisis , Calmodulina/análisis , Calmodulina/química , Calmodulina/metabolismo , Células A549 , Límite de Detección , Técnicas Biosensibles/métodos , Ionomicina/farmacologíaRESUMEN
This study aimed to identify for the first time protein biomarkers of meat quality traits from Longissimus thoracis (LT) muscle of goats (Capra hircus). Male goats of similar age and weight reared under extensive rearing conditions were used to relate the LT muscle proteome with multiple meat quality traits. The early post-mortem muscle proteome analyzed using label-free proteomics was compared among three texture clusters built using hierarchical clustering analysis. Twenty-five proteins were differentially abundant and their mining using bioinformatics revealed three major biological pathways to be involved: 10 muscle structure proteins (MYL1, MYL4, MYLPF, MYL6B, MYH1, MYH2, ACTA1, ACTBL2, FHL1 and MYOZ1); 6 energy metabolism proteins (ALDOA, PGAM2, ATP5F1A, GAPDH, PGM1 and ATP5IF1), and two heat shock proteins: HSPB1 (small) and HSPA8 (large). Seven other miscellaneous proteins belonging to pathways such as regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding were further identified to play a role in the variability of goat meat quality. The differentially abundant proteins were correlated with the goat meat quality traits in addition to multivariate regression models built to propose the first regression equations of each quality trait. This study is the first to highlight in a multi-trait quality comparison the early post-mortem changes in the goat LT muscle proteome. It also evidenced the mechanisms underpinning the development of several quality traits of interest in goat meat production along the major biochemical pathways at interplay. SIGNIFICANCE: The discovery of protein biomarkers in the field of meat research is an emerging topic. In the case of goat meat quality, very few studies using proteomics have been conducted with the aim of proposing biomarkers. Therefore, this study is the first to quest for biomarkers of goat meat quality using label-free shotgun proteomics with a focus on multiple quality traits. We identified the molecular signatures underlying goat meat texture variation, which were found to belong to muscle structure and related proteins, energy metabolism and heat shock proteins along with other proteins involved in regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding. We further evaluated the potential of the candidate biomarkers to explain meat quality using the differentially abundant proteins by means of correlation and regression analyses. The results allowed the explanation of the variation in multiple traits such as pH, color, water-holding capacity, drip and cook losses traits and texture.
Asunto(s)
Proteoma , Proteómica , Masculino , Animales , Proteoma/metabolismo , Proteómica/métodos , Calmodulina/análisis , Calmodulina/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Choque Térmico/metabolismo , Carne/análisis , Biomarcadores/análisis , Cabras/metabolismo , ARN de Transferencia/análisis , ARN de Transferencia/metabolismo , Músculo Esquelético/químicaRESUMEN
Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is the most abundant protein in excitatory synapses and is central to synaptic plasticity, learning and memory. It is activated by intracellular increases in calcium ion levels and triggers molecular processes necessary for synaptic plasticity. CaMKII phosphorylates numerous synaptic proteins, thereby regulating their structure and functions. This leads to molecular events crucial for synaptic plasticity, such as receptor trafficking, localization and activity; actin cytoskeletal dynamics; translation; and even transcription through synapse-nucleus shuttling. Several new tools affording increasingly greater spatiotemporal resolution have revealed the link between CaMKII activity and downstream signalling processes in dendritic spines during synaptic and behavioural plasticity. These technologies have provided insights into the function of CaMKII in learning and memory.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calmodulina , Humanos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/análisis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/análisis , Calmodulina/metabolismo , Calcio/metabolismo , Actinas/análisis , Actinas/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/metabolismo , HipocampoRESUMEN
Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.
Asunto(s)
Técnicas Biosensibles , Calmodulina/análisis , Diseño de Equipo , Tecnología de Fibra Óptica , Oro , Fibras Ópticas , Refractometría , Resonancia por Plasmón de SuperficieRESUMEN
All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our lab's efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.
Asunto(s)
Técnicas Biosensibles , Calcineurina/análisis , Calmodulina/análisis , Fluorescencia , Diana Mecanicista del Complejo 1 de la Rapamicina/análisis , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.
Asunto(s)
Aptámeros de Péptidos/química , Calmodulina/análisis , Proteínas Inmovilizadas/química , Secuencia de Aminoácidos , Aptámeros de Péptidos/genética , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica , Evolución Molecular Dirigida , Proteínas Inmovilizadas/genética , Límite de Detección , Polímeros/química , Ingeniería de ProteínasRESUMEN
Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H2O2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.
Asunto(s)
Metionina/metabolismo , Péptidos/química , Proteínas/química , Espectrometría de Masas en Tándem/métodos , Apoproteínas/análisis , Apoproteínas/química , Calmodulina/análisis , Calmodulina/química , Peróxido de Hidrógeno/química , Radical Hidroxilo/química , Metionina/análisis , Metionina/química , Mioglobina/análisis , Mioglobina/química , Oxidación-Reducción , Péptidos/análisis , Huella de Proteína , Procesamiento Proteico-Postraduccional , Proteínas/análisis , Reproducibilidad de los ResultadosRESUMEN
The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels.
Asunto(s)
Calmodulina/química , Calmodulina/genética , Schistosoma mansoni/genética , Secuencia de Aminoácidos , Animales , Calmodulina/análisis , Humanos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.
Asunto(s)
Compuestos Macrocíclicos/química , Factor de Necrosis Tumoral alfa/análisis , Calmodulina/análisis , Anhidrasa Carbónica IX/análisis , Anhidrasa Carbónica IX/metabolismo , Peroxidasa de Rábano Silvestre/análisis , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Microscopía Fluorescente , Orosomucoide/análisis , Antígeno Prostático Específico/análisis , Albúmina Sérica Humana/análisis , Tanquirasas/análisis , Tanquirasas/metabolismoRESUMEN
OBJECTIVE: Cupping therapy has a long history in traditional medicine especially in Asian countries. It was controversial whether cupping induced blisters are beneficial to healing effects, and the formation and content in the blisters remain unexplored. We aimed to identify and compare the molecular components of the blister fluid from the cupping therapy and the scalds to explore the necessary of inducing cupping induced blisters. METHODS: Fluid sample of blisters from fifteen patients receiving cupping therapy (Cupping group) and scald burns (Scald group) were collected in this study. Proteins from the blisters were separated by two-dimensional electrophoresis (2D-gel) and further analyzed by mass spectrometry. In addition, the changes in particular proteins were confirmed by Western blotting. RESULTS: The protein components are significantly different between blister from cupping therapy and scalds. The immune responses, oxidative stress and metabolic related proteins (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase and tetranectin) were increased, whereas the hemoglobin subunit beta was decreased in the Cupping group compared with the Scald group. CONCLUSIONS: Cupping induced blisters contain several proteins which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping.
Asunto(s)
Vesícula , Líquidos Corporales/química , Terapias Complementarias , Medicina Tradicional China , Proteoma , Vesícula/inmunología , Vesícula/metabolismo , Venodisección , Calmodulina/análisis , Electroforesis en Gel Bidimensional , Hemopexina/análisis , Humanos , Inmunoglobulinas/análisis , Proteoma/análisis , Proteoma/metabolismo , ProteómicaRESUMEN
In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight ( Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/análisis , Calmodulina/análisis , Supervivencia Celular , Escherichia coli/química , Conformación Proteica , Proteómica/métodos , Proteínas Recombinantes/análisisRESUMEN
We present the first cryptophane-based "turn-on" 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.
Asunto(s)
Técnicas Biosensibles , Calmodulina/análisis , Compuestos Policíclicos/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Isótopos de XenónRESUMEN
Calmodulin (CaM) is a Ca2+-binding protein that plays a role in several Ca2+ signaling pathways, which dynamically regulates the activities of hundreds of proteins. The ice alga Chlamydomonas sp. ICE-L, which has the ability to adapt to extreme polar conditions, is a crucial primary producer in Antarctic ecosystem. This study hypothesized that Cam helps the ICE-L to adapt to the fluctuating conditions in the polar environment. It first verified the overall length of Cam, through RT-PCR and RACE-PCR, based on partial Cam transcriptome library of ICE-L. Then, the nucleotide and predicted amino acid sequences were, respectively, analyzed by various bioinformatics approaches to gain more insights into the computed physicochemical properties of the CaM. Potential involvements of Cam in responding to certain stimuli (i.e., UVB radiation, high salinity, and temperature) were investigated by differential expression, measuring its transcription levels by means of quantitative RT-PCR. Results showed that CaM was indeed inducible and regulated by high UVB radiation, high salinity, and nonoptimal temperature conditions. Different conditions had different expression tendencies, which provided an important basis for investigating the adaptation mechanism of Cam in ICE-L.
Asunto(s)
Calmodulina/análisis , Calmodulina/genética , Chlamydomonas/enzimología , Perfilación de la Expresión Génica , Regiones Antárticas , Calmodulina/química , Chlamydomonas/efectos de los fármacos , Chlamydomonas/genética , Chlamydomonas/efectos de la radiación , Clonación Molecular , Biología Computacional , Presión Osmótica , Reacción en Cadena de la Polimerasa , Salinidad , Temperatura , Rayos UltravioletaRESUMEN
High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at â¼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.
Asunto(s)
Encéfalo/citología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas/fisiología , Animales , Encéfalo/fisiología , Calmodulina/análisis , Calmodulina/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred.
Asunto(s)
Calcio/química , Calmodulina/química , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Escherichia coli/química , Escherichia coli/citología , Sitios de Unión , Calmodulina/análisis , Iones/químicaRESUMEN
Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.
Asunto(s)
Algoritmos , Calmodulina/análisis , Plectina/análisis , Calmodulina/química , Reactivos de Enlaces Cruzados/química , Bases de Datos de Proteínas , Humanos , Plectina/química , Succinimidas/química , Espectrometría de Masas en TándemRESUMEN
The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of <100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca(2+). Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from <0.01µm(2)s(-1) to ~5µm(2) s(-1), whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of >10µm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.
Asunto(s)
Calmodulina/análisis , Imagen Individual de Molécula , Transporte Biológico , Señalización del Calcio , Carbocianinas , Difusión , Ácido Egtácico/análogos & derivados , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes , Células HEK293 , Humanos , Microinyecciones , Unión Proteica , Fracciones Subcelulares/químicaRESUMEN
Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM) binding, indicating a potential role in the plant calcium signalling pathway. To provide a better understanding of the IQ motif-mediated interaction between AtBAG5 and CaM, the two proteins were expressed and purified separately and then co-crystallized together. Diffraction-quality crystals of the complex were grown using the sitting-drop vapour-diffusion technique from a condition consisting of 0.1â M Tris-HCl pH 8.5, 2.5â M ammonium sulfate. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.56, b = 74.89, c = 117.09â Å. X-ray diffraction data were recorded to a resolution of 2.5â Å from a single crystal using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, a Matthews coefficient of 2.44â Å(3)â Da(-1) was calculated, corresponding to a solvent content of approximately 50%.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis , Calmodulina/química , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Calmodulina/análisis , Calmodulina/genética , Cristalización , Cristalografía por Rayos X/métodos , Datos de Secuencia MolecularRESUMEN
Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.
Asunto(s)
Calmodulina/análisis , Calmodulina/química , Reactivos de Enlaces Cruzados/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bovinos , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , Estructura Secundaria de ProteínaRESUMEN
Calneuron-1 and -2 are members of the neuronal calcium-binding protein family (nCaBP). They are transmembrane Calmodulin-like EF-hand Ca(2+)-sensors, and a function in the control of Golgi-to-plasma membrane vesicle trafficking has been assigned to both proteins. In this paper, we describe the distribution of Calneuron-1 in rat and human brains. We show that Calneuron-1 is ubiquitously expressed in all brain regions examined. The protein is most abundant in Purkinje cells of the cerebellum and principal neurons of the cortex and limbic brain whereas no expression in glial cells is apparent. In addition, we identify two novel splice isoforms of Calneuron-1 with extended N-termini. These isoforms are particular abundant in the cerebellum. Taken together, these data set grounds for a better understanding of the cellular function of Calneurons.