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Cloning
https://doi.org/10.1016/S0378-1119(98)00433-8
A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.
Comments: Article useful to confirm the design of the GFP-fused protein expression systems.
https://doi.org/10.1128/AEM.01072-09
Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.
Comments: Article useful to confirm the design of the GFP-fused protein expression systems.
https://doi.org/10.1266/ggs.89.169
Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.
Comments: Article useful to confirm the design of the GFP-fused protein expression systems.
https://doi.org/10.1046/j.1365-313x.1998.00304.x
The C-terminus of mouse talin (amino acids 2345-2541) is responsible for all of the protein’s f-actin binding capacity. Unlike full-length talin, the C-terminal f-actin binding domain is unable to nucleate actin polymerization. We have found that transient and stable expression of the talin actin-binding domain fused to the C-terminus of the green fluorescent protein (GFP-mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function. Transiently expressed GFP-mTn co-localized with rhodamine-phalloidin in permeabilized tobacco BY-2 suspension cells, showing that the fusion protein can specifically label the plant actin cytoskeleton. Constitutive expression of GFP-mTn in transgenic Arabidopsis thaliana plants visualized actin filaments in all examined tissues with no apparent effects on plant morphology or development at any stage during the life cycle. This demonstrates that in a number of different cell types GFP-mTn can serve as a non-invasive marker for the actin cytoskeleton. Confocal imaging of GFP-mTn labeled actin filaments was employed to reveal novel information on the in vivo organization of the actin cytoskeleton in transiently transformed, normally elongating tobacco pollen tubes.
Comments: Article useful to confirm the design of the GFP-fused protein expression systems.
De novo design
https://doi.org/10.1038/nature19946
There are 20^200 possible amino-acid sequences for a 200-residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding. Computational methodology has advanced to the point that a wide range of structures can be designed from scratch with atomic-level accuracy. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnology.
Comments: Description of the protein sequence space and the emergence of new folding design.
https://doi.org/10.1038/s41586-024-08271-6
Intrinsically disordered proteins can bind via the formation of highly disordered protein complexes without the formation of three-dimensional structure1. Most naturally occurring proteins are levorotatory (L)—that is, made up only of L-amino acids—imprinting molecular structure and communication with stereochemistry2. By contrast, their mirror-image dextrorotatory (D)-amino acids are rare in nature. Whether disordered protein complexes are truly independent of chiral constraints is not clear. Here, to investigate the chiral constraints of disordered protein–protein interactions, we chose as representative examples a set of five interacting protein pairs covering the disorder–order continuum. By observing the natural ligands and their stereochemical mirror images in free and bound states, we found that chirality was inconsequential in a fully disordered complex. However, if the interaction relied on the ligand undergoing extensive coupled folding and binding, correct stereochemistry was essential. Between these extremes, binding could be observed for the D-ligand with a strength that correlated with disorder in the final complex. These findings have important implications for our understanding of the molecular processes that lead to complex formation, the use of D-peptides in drug discovery and the chemistry of protein evolution of the first living entities on Earth.
Comments: This is a great example of what can be done with CD for the characterization of left-handed structures.
Data processing
https://doi.org/10.1107/S1600576724009506
Analytical absorption corrections are employed in scaling diffraction data for highly absorbing samples, such as those used in long-wavelength crystallography, where empirical corrections pose a challenge. AnACor2.0 is an accelerated software package developed to calculate analytical absorption corrections. It accomplishes this by ray-tracing the paths of diffracted X-rays through a voxelized 3D model of the sample. Due to the computationally intensive nature of ray-tracing, the calculation of analytical absorption corrections for a given sample can be time consuming. Three experimental datasets (insulin at lambda=3.10 A, thermolysin at lambda= 3.53 A and thaumatin at lambda= 4.13 A) were processed to investigate the effectiveness of the accelerated methods in AnACor2.0. These methods demonstrated a maximum reduction in execution time of up to 175x compared with previous methods. As a result, the absorption factor calculation for the insulin dataset can now be completed in less than 10 s. These acceleration methods combine sampling, which evaluates subsets of crystal voxels, with modifications to standard ray-tracing. The bisection method is used to find path lengths, reducing the complexity from O(n) to O(log2 n). The gridding method involves calculating a regular grid of diffraction paths and using interpolation to find an absorption correction for a specific reflection. Additionally, optimized and specifically designed CUDA implementations for NVIDIA GPUs are utilized to enhance performance. Evaluation of these methods using simulated and real datasets demonstrates that systematic sampling of the 3D model provides consistently accurate results with minimal variance across different sampling ratios. The mean difference of absorption factors from the full calculation (without sampling) is at most 2%. Additionally, the anomalous peak heights of sulfur atoms in the Fourier map show a mean difference of only 1% compared with the full calculation. This research refines and accelerates the process of analytical absorption corrections, introducing innovative sampling and computational techniques that significantly enhance efficiency while maintaining accurate results.
Comments: Introduction of an absorption correction algorithm that could be useful for noise-reduction during data integration and diffraction simulation.
https://doi.org/10.1107/S1600577514015847
A new approach is proposed for measuring structural dynamics in materials from multi-speckle scattering patterns obtained with partially coherent X-rays. Coherent X-ray scattering is already widely used at high-brightness synchrotron lightsources to measure dynamics using X-ray photon correlation spectroscopy, but in many situations this experimental approach based on recording long series of images (i.e. movies) is either not adequate or not practical. Following the development of visible-light speckle visibility spectroscopy, the dynamic information is obtained instead by analyzing the photon statistics and calculating the speckle contrast in single scattering patterns. This quantity, also referred to as the speckle visibility, is determined by the properties of the partially coherent beam and other experimental parameters, as well as the internal motions in the sample (dynamics). As a case study, Brownian dynamics in a low-density colloidal suspension is measured and an excellent agreement is found between correlation functions measured by X-ray photon correlation spectroscopy and the decay in speckle visibility with integration time obtained from the analysis presented here.
Comments: Description of an approach to study the dynamics that exist in diffraction speckles. This could be used to study the radiation damage within protein crystals.
Detectors
https://doi.org/10.1016/S0378-1119(98)00433-8
Lead-based relaxor ferroelectrics exhibit giant piezoelectric properties owing to their heterogeneous structures. The average and local structures measured by single-crystal X-ray diffraction under DC and AC electric fields are reviewed in this article. The position-dependent local lattice strain and the distribution of polar nanodomains and nanoregions show strong electric field dependence, which contributes to the giant piezoelectric properties.
Comments: Article from Aoyagi that used the Timepix detector from CERN.
https://doi.org/10.1107/S160057752400986X
We report on recent developments that enable megahertz hard X-ray phase contrast imaging (MHz XPCI) experiments at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL facility (EuXFEL). We describe the technical implementation of the key components, including an MHz fast camera and a modular indirect X-ray microscope system based on fast scintillators coupled through a high-resolution optical microscope, which enable full-field X-ray microscopy with phase contrast of fast and irreversible phenomena. The image quality for MHz XPCI data showed significant improvement compared with a pilot demonstration of the technique using parallel beam illumination, which also allows access to up to 24 keV photon energies at the SPB/SFX instrument of the EuXFEL. With these developments, MHz XPCI was implemented as a new method offered for a broad user community (academic and industrial) and is accessible via standard user proposals. Furthermore, intra-train pulse diagnostics with a high few-micrometre spatial resolution and recording up to 128 images of consecutive pulses in a train at up to 1.1 MHz repetition rate is available upstream of the instrument. Together with the diagnostic camera upstream of the instrument and the MHz XPCI setup at the SPB/SFX instrument, simultaneous two-plane measurements for future beam studies and feedback for machine parameter tuning are now possible.
Comments: development of a camera system with MHz time resolution using a FastCam and a scintillator CRY-60 with sufficiently short decay time.
https://doi.org/10.1107/S1600577524010567
Coherent diffraction microscopy (CDM) is a robust direct imaging method due to its unique 2D/3D phase retrieval capacity. Nonetheless, its resolution faces limitations due to a diminished signal-to-noise ratio (SNR) in high-frequency regions. Addressing this challenge, X-ray ensemble diffraction microscopy (XEDM) emerges as a viable solution, ensuring an adequate SNR in high-frequency regions and effectively surmounting resolution constraints. In this article, two experiments were conducted to underscore XEDM’s superior spatial resolution capabilities. These experiments employed 55 nm-sized silicon–gold nanoparticles (NPs) and 19 nm-sized nodavirus-like particles (NV-LPs) on the coherent X-ray scattering beamline of the Taiwan Photon Source. The core–shell density distribution of the silicon–gold NPs was successfully obtained with a radial resolution of 3.4 nm per pixel, while NV-LPs in solution were reconstructed at a radial resolution of 1.3 nm per pixel. The structural information was directly retrieved from the diffraction intensities without prior knowledge and was subsequently confirmed through transmission electron microscopy.
Comments: use of the Eiger for X-ray ensemble diffraction microscopy (XEDM) in Taiwan.
In vivo MX
https://doi.org/10.1038/s41586-024-08203-4
Although separation is entropically unfavourable, it is often essential for our life1,2. The separation of very similar macromolecules such as deoxyribonucleic acids (DNAs) and their single nucleotide variants is difficult but holds great advantage for the progress of life science3. Here we report that a particular liquid–liquid phase separation (LLPS) at a solid–liquid interface led to the partitioning of DNAs with nearly identical structures. We found this intriguing phenomenon when we did drop-casting onto a glass plate an aqueous ammonium sulfate dispersion of phase-separated droplets comprising a homogeneous mixture of poly(ethylene glycol) (PEG) samples with different termini. Even when the molecular weights of their PEG parts were identical to each other, terminally different PEGs spread competitively at the solid–liquid interface and partitioned into micrometre-scale concentric circles. We found that this competitive spreading was induced by an ammonium sulfate layer spontaneously formed on the glass surface. We successfully extended the above mechanism to partitioning a mixture of nearly identical DNAs into concentric circles followed by their selective extraction using the salting-in effect. We could isolate a human cancer-causing single nucleotide variant in 97% purity from its 1:1 mixture with the original DNA.
Comments: The application could be used to image and understand better what is happening inside living cells during in vivo crystallization phenomena.
https://www.ncbs.res.in/sitefiles/skumarpaper.pdf
Understanding the structural heterogeneity inherent in the process of amyloid fibril formation is an important goal of protein aggregation studies. Structural heterogeneity in amyloid fibrils formed by a protein manifests itself in fibrils varying in internal structure and external appearance, and may originate from molecular level variations in the internal structure of the cross-β motif. Amyloid fibril formation commences from partially structured conformations of a protein, and in many cases, proceeds via pre-fibrillar aggregates (spherical oligomers and/or protofibrils). It now appears that structural heterogeneity is prevalent in the partially structured conformations as well as in the pre-fibrillar aggregates of proteins. Amyloid fibril formation may therefore potentially commence from many precursor states, and amyloid fibril polymorphism might be the consequence of the utilization of distinct nucleation and elongation mechanisms. This review examines the current understanding of the structural heterogeneity seen in amyloid fibril formation reactions, and describes how an understanding of the initial and intermediate stages of amyloid fibril formation reactions can provide an insight into the structural heterogeneity seen in mature fibrils.
Comments: Introduction of the general methodology to detect amyloid formation.
https://doi.org/10.1016/j.tibs.2020.05.011
Many methods have been developed to examine the bulk properties and underlyingmolecular interactions of biomolecular condensates that form through liquid–liquid phase separation (LLPS). Phase separation assays using bright-field imaging or fluorescence microscopy track droplets over time and can assess the effect of various conditions on LLPS, measure droplet formation and dissolution kinetics, and test the miscibility of differentially labeled molecules. Fluorescence recovery after photobleaching (FRAP) and microrheology by particle tracking probe the diffusion of molecules within droplets, while droplet fusion assays, right angle imaging, and surface wetting determine droplet surface tension.
Comments: Introduction of various methods used to study the LLPS.
https://doi.org/10.1038/s41392-021-00678-1
Emerging evidence suggests that liquid–liquid phase separation (LLPS) represents a vital and ubiquitous phenomenon underlying the formation of membraneless organelles in eukaryotic cells (also known as biomolecular condensates or droplets). Recent studies have revealed evidences that indicate that LLPS plays a vital role in human health and diseases. In this review, we describe our current understanding of LLPS and summarize its physiological functions. We further describe the role of LLPS in the development of human diseases. Additionally, we review the recently developed methods for studying LLPS. Although LLPS research is in its infancy—but is fast-growing—it is clear that LLPS plays an essential role in the development of pathophysiological conditions. This highlights the need for an overview of the recent advances in the field to translate our current knowledge regarding LLPS into therapeutic discoveries.
Comments: Summary of the implication of LLPS in human diseases.
https://doi.org/10.1038/s41580-020-00326-6
Biomolecular condensates are membraneless intracellular assemblies that often form via liquid−liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.
Comments: Introduction of implication of LLPS in human diseases.
https://dx.doi.org/10.1021/acs.jpclett.0c02567
Increasing experiments suggest that amyloid peptides can undergo liquid−liquid phase separation (LLPS) before the formation of amyloid fibrils. However, the exact role of LLPS in amyloid aggregation at the molecular level remains elusive. Here, we investigated the LLPS and amyloid fibrillization of a coarse-grained peptide, capable of capturing fundamental properties of amyloid aggregation over a wide range of concentrations in molecular dynamics simulations. On the basis of the Flory−Huggins theory of polymer solutions, we determined the binodal and spinodal concentrations of LLPS in the low-concentration regime, ϕBL and ϕSL, respectively. Only at concentrations above ϕBL, peptides formed metastable or stable oligomers corresponding to the high-density liquid phase (HDLP) in LLPS, out of which the nucleated conformational conversion to fibril seeds occurred. Below ϕSL, the HDLP was metastable and transient, and the subsequent fibrillization process followed the traditional nucleation and elongation mechanisms. Only above ϕSL, the HDLP became stable, and the initial fibril nucleation and growth were governed by the high local peptide concentrations. The predicted saturation of amyloid aggregation half-times with increasing peptide concentration to a constant, instead of the traditional power-law scaling to zero, was confirmed by simulations and by a thioflavin-T kinetic assay and the transmission electron microscopy of islet amyloid polypeptide (IAPP) aggregation. Our study provides a unified picture of amyloid aggregation for a wide range of concentrations within the framework of LLPS, which may help us better understand the etiology of amyloid diseases, where the amyloid protein concentration can vary by ∼9 orders of magnitude depending on the organ location and facilitate the engineering of novel amyloid-based functional materials.
Comments: Implications of LLPS in Amyloid aggregation.
https://doi.org/10.1016/j.jbc.2023.102926
Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer’s disease, but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid–liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of SDS. Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that the droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (−)-epigallocatechin gallate. Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of Alzheimer’s disease.
Comments: Introduction of the implication of LLPS in amyloid formation
https://doi.org/10.3390/molecules27144588
The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.
Comments: Introduction of the implication of LLPS in Amyloid formation.
https://doi.org/10.1186/s43593-023-00048-0
The accumulation and deposition of amyloid can cause a variety of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. The degradation or clearance of this accumulation is currently the most widely accepted therapeutic strategy for intervention in these pathologies. Our study on amyloid-β (Aβ) oligomers in vitro revealed that high-frequency terahertz (THz) waves at a specific frequency of 34.88 THz could serve as a physical, efficient, nonthermal denaturation technique to delay the fibrotic process by 80%, as monitored by a thioflavine T (ThT) binding assay and Fourier transform infrared (FTIR) spectroscopy. Additionally, THz waves of this frequency have been shown to have no side effects on normal cells, as confirmed by cell viability and mitochondrial membrane potential assays. Furthermore, molecular dynamic (MD) simulations revealed that the THz waves could resonate with Aβ fibrils, disrupting the dense conformation by breaking the β-sheet structure and promoting the formation of abundant coil and bend structures. This study uses the amyloid of Aβ as an example, and the results will further guide interventions for the accumulation of other amyloids, which may provide new ideas for the remission of related diseases.
Comments: Introduction of a potential solution to treat Amyloid formation.
https://doi.org/10.1002/chem.202400277
Amyloid plaques are a major pathological hallmark involved in Alzheimer’s disease and consist of deposits of the amyloid-β peptide (Aβ). The aggregation process of Aβ is highly complex, which leads to polymorphous aggregates with different structures. In addition to aberrant aggregation, Aβ oligomers can undergo liquid-liquid phase separation (LLPS) and form dynamic condensates. It has been hypothesized that these amyloid liquid droplets affect and modulate amyloid fibril formation. In this review, we briefly introduce the relationship between stress granules and amyloid protein aggregation that is associated with neurodegenerative diseases. Then we highlight the regulatory role of LLPS in Aβ aggregation and discuss the potential relationship between Aβ phase transition and aggregation. Furthermore, we summarize the current structures of Aβ oligomers and amyloid fibrils, which have been determined using nuclear magnetic resonance (NMR) and cryoelectron microscopy (cryo-EM). The structural variations of Aβ aggregates provide an explanation for the different levels of toxicity, shed light on the aggregation mechanism and may pave the way towards structure-based drug design for both clinical diagnosis and treatment.
Comments; Review reporting the mechanisms of aggregation of Amyloid fibers.
https://doi.org/10.1093/jb/mvad056
Membraneless organelles formed via the self-assembly of intrinsically disordered proteins (IDPs) play a crucial role in regulating various physiological functions. Elucidating the mechanisms behind IDP self-assembly is of great interest not only from a biological perspective but also for understanding how amino acid mutations in IDPs contribute to the development of neurodegenerative diseases and other disorders. Currently, two proposed mechanisms explain IDP self-assembly: (1) the sticker-and-spacer framework, which considers amino acid residues as beads to simulate the intermolecular interactions, and (2) the cross-β hypothesis (Graphical Abstract), which focuses on the β-sheet interactions between the molecular surfaces constructed by multiple residues. This review explores the advancement of new models that provide higher-resolution insights into the IDP self-assembly mechanism based on new findings obtained from structural studies of IDPs.
Comments: Review of various studies involved in protein aggregation.