miniaturized-sample-preparation-for-transmission-electron-microscopy

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

identification-functional-protein-regions-through-chimeric-protein

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the interaction of MRP with CaM.

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy.

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution. Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer.

Micromechanical cantilever arrays are used to measure time-resolved adsorption of tiny masses based on protein-ligand interactions. Here, streptavidin-biotin interactions are investigated in a physiological environment. A measurement method is introduced using higher flexural modes of a silicon cantilever in order to enhance the sensitivity of mass detection. Modeling the cantilever vibration in liquid allows the measurement of absolute mass changes. We show time-resolved mass adsorption of final 7+/-0.7 ng biotinylated latex beads. The sensitivity obtained is about 2.5 pg/Hz measuring at a center frequency of 750 kHz.

Micromechanical mass sensors for biomolecular detection in a physiological environment.

Bacteriorhodopsin proteoliposomes were used as a model system to explore the applicability of micromechanical cantilever arrays to detect conformational changes in membrane protein patches. The three main results of our study concern: 1), reliable functionalization of micromechanical cantilever arrays with proteoliposomes using ink jet spotting; 2), successful detection of the prosthetic retinal removal (bleaching) from the bacteriorhodopsin protein by measuring the induced nanomechanical surface stress change; and 3), the quantitative response thereof, which depends linearly on the amount of removed retinal. Our results show this technique to be a potential tool to measure membrane protein-based receptor-ligand interactions and conformational changes.

Conformational change of bacteriorhodopsin quantitatively monitored by microcantilever sensors.

Amphiphilic di- and triblock copolymers having different hydrophilic-to-hydrophobic block length ratio were synthesized using ATRP. The self-assembly behavior of these AB and ABA block copolymers consisting of poly(n-butyl methacrylate) (B) and poly(2,2-(dimethylaminoethyl methacrylate) (A) was investigated using a combination of dynamic light scattering, negative-stain transmission electron microscopy, cryoelectron microscopy, and atomic force microscopy. Two populations of self-organized structures in aqueous solution, micelles and compound micelles, were detected for diblock copolymers. Triblock copolymers assembled into vesicular structures of uniform sizes. Furthermore it was found that these vesicles tended to compensate the high curvature by additional organization of the polymer chains outside of the membrane. The chain hydrophilicity of the polymers appeared to have a critical impact on the self-assembly response toward temperature change. The self-reorganization of the polymers at different temperatures is discussed.

Self-organization behavior of methacrylate-based amphiphilic di- and triblock copolymers.

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

Comprehensive characterization of molecular interactions based on nanomechanics.

Polymers are often used to modify surface properties to control interfacial processes. Their sensitivity to solvent conditions and ability to undergo conformational transitions makes polymers attractive in tailoring surface properties with specific functionalities leading to applications in diverse areas ranging from tribology to colloidal stability and medicine. A key example is polyethylene glycol (PEG), which is widely used as a protein-resistant coating given its low toxicity and biocompatibility. We report here a microcantilever-based sensor for the in situ characterization of PEG monolayer formation on Au using the "grafting to" approach. Moreover, we demonstrate how microcantilevers can be used to monitor conformational changes in the grafted PEG layer in different solvent conditions. This is supported by atomic force microscope (AFM) images and force-distance curve measurements of the microcantilever chip surface, which show that the grafted PEG undergoes a reversible collapse when switching between good and poor solvent conditions, respectively.

Sensing surface PEGylation with microcantilevers.

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM. 

Single-cell lysis for visual analysis by electron microscopy.

A microcantilever based method for fluid viscosity and mass density measurements with high temporal resolution and microliter sample consumption is presented. Nanomechanical cantilever vibration is driven by photothermal excitation and detected by an optical beam deflection system using two laser beams of different wavelengths. The theoretical framework relating cantilever response to the viscosity and mass density of the surrounding fluid was extended to consider higher flexural modes vibrating at high Reynolds numbers. The performance of the developed sensor and extended theory was validated over a viscosity range of 1-20 mPa·s and a corresponding mass density range of 998-1176 kg/m(3) using reference fluids. Separating sample plugs from the carrier fluid by a two-phase configuration in combination with a microfluidic flow cell, allowed samples of 5 μL to be sequentially measured under continuous flow, opening the method to fast and reliable screening applications. To demonstrate the study of dynamic processes, the viscosity and mass density changes occurring during the free radical polymerization of acrylamide were monitored and compared to published data. Shear-thinning was observed in the viscosity data at higher flexural modes, which vibrate at elevated frequencies. Rheokinetic models allowed the monomer-to-polymer conversion to be tracked in spite of the shear-thinning behavior, and could be applied to study the kinetics of unknown processes. 

Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators.

New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis.

openBEB: open biological experiment browser for correlative measurements.

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed. 

Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics".

The complex biology of KIT cells in the heart.

The pleiotropic interleukin-6 (IL-6)-type cytokine oncostatin M (OSM) signals in multiple cell types, affecting processes such as cell differentiation, hematopoiesis, and inflammation. In humans, OSM exerts its effects through activation of either of two different heterodimeric receptor complexes, formed by glycoprotein 130 (gp130) and either OSM receptor (OSMR) or leukemia inhibitory factor receptor (LIFR). In contrast, the mouse OSM orthologue acts mainly through dimers containing OSMR and gp130 and shows limited activity through mouse LIFR. Despite their structural similarity, neither human nor mouse OSM signal through the other species' OSMR. The molecular basis for such species-specific signaling, however, remains poorly understood. To identify key molecular features of OSM that determine receptor activation in humans and mice, we generated chimeric mouse-human cytokines. Replacing regions within binding site III of murine OSM with the human equivalents showed that the cytokine's AB loop was critical for receptor selection. Substitutions of individual amino acids within this region demonstrated that residues Asn-37, Thr-40, and Asp-42 of the murine cytokine were responsible for limited LIFR activation and absence of human OSMR/LIFR signaling. In human OSM, Lys-44 appeared to be the main residue preventing mouse OSMR activation. Our data reveal that individual amino acids within the AB loop of OSM determine species-specific activities. These mutations might reflect a key step in the evolutionary process of this cytokine, in which receptor promiscuity gives way to ligand-receptor specialization.

The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice.

Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1 CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.

ArticleTotal : 2	Year
Publication title Cited by 11	2018
Publication title	2019

Article	Year
Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 10792048	2000
The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer. EMBO reports\| PubMed ID: 11265760	2000
Micromechanical mass sensors for biomolecular detection in a physiological environment. Physical review. E, Statistical, nonlinear, and soft matter physics\| PubMed ID: 16241482	2005
Conformational change of bacteriorhodopsin quantitatively monitored by microcantilever sensors. Biophysical journal\| PubMed ID: 16443650	2006
Self-organization behavior of methacrylate-based amphiphilic di- and triblock copolymers. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 17949024	2007
Comprehensive characterization of molecular interactions based on nanomechanics. PloS one\| PubMed ID: 18978938	2008
Sensing surface PEGylation with microcantilevers. Beilstein journal of nanotechnology\| PubMed ID: 21977390	2010
Single-cell lysis for visual analysis by electron microscopy. Journal of structural biology\| PubMed ID: 23816812	2013
Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators. Analytical chemistry\| PubMed ID: 23905589	2013
openBEB: open biological experiment browser for correlative measurements. BMC bioinformatics\| PubMed ID: 24666611	2014
Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy. Analytical chemistry\| PubMed ID: 24773394	2014
Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts. Journal of structural biology\| PubMed ID: 27864160	2017
Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics". Proteomics\| PubMed ID: 29441686	2018
The complex biology of KIT cells in the heart. Nature reviews. Cardiology\| PubMed ID: 29802343	2018
The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice. The Journal of biological chemistry\| PubMed ID: 30373773	2018
Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement. Nature communications\| PubMed ID: 30451828	2018

Thomas Braun

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