Investigation of the Efficacy of Lanthanoid Heavy Metal Acetates as Electron Staining Reagents for Biomembrane Vesicles.

Transmission electron microscopy (TEM) has revolutionized our understanding of protein structures by enabling atomic-resolution visualization without the need for crystallography, thanks to advancements in cryo-TEM and single particle analysis methods. However, conventional electron microscopy remains relevant for studying stained samples, as it allows the practical determination of optimal conditions through extensive experimentation. TEM also facilitates the examination of supramolecular complexes encompassing proteins, lipids, and nucleic acids. In this study, we investigated the applicability of lanthanoid reagents as electron-staining alternatives to uranyl acetate, which is globally regulated as a nuclear fuel material. We focus on a model biomembrane vesicle system, the chromatophores from the purple photosynthetic eubacterium Rhodospirillum rubrum, which integrate proteins and lipids. Through density distribution analysis of electron micrographs, we evaluated the efficacy of various lanthanoid acetates and found that triacetates of neodymium, samarium, and gadolinium exhibited similar staining effectiveness to uranyl acetate. Additionally, triacetates of praseodymium, erbium, and lutetium, followed by europium show promising results as secondary candidates. Our findings suggest that lanthanoid transition heavy metal acetates could serve as viable alternatives for electron staining in TEM, offering potential advantages over uranyl acetate.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Intermolecular interactions at early stage of protein/detergent particle association induced by salt/polyethylene glycol mixtures.

Mixtures of neutral salts and polyethylene glycol are used for various purposes in biological studies. Although the effects of each component of the mixtures are theoretically well investigated, comprehension of their integrated effects remains insufficient. In this work, their roles and effects as a precipitant were clarified by studying dependence of precipitation curves on salt concentration for integral membrane protein/detergent particles of different physicochemical properties. The dependence of precipitation curves was reasonably related to intermolecular interactions among relevant molecules such as protein, detergent and polyethylene glycol by considering their physicochemical properties. The obtained relationships are useful as basic information to learn the early stage of biological macromolecular associations.

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions.

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.

Crystal structure of a ten-amino acid protein.

What is the smallest protein? This is actually not such a simple question to answer, because there is no established consensus among scientists as to the definition of a protein. We describe here a designed molecule consisting of only 10 amino acids. Despite its small size, its essential characteristics, revealed by its crystal structure, solution structure, thermal stability, free energy surface, and folding pathway network, are consistent with the properties of natural proteins. The existence of this kind of molecule deepens our understanding of proteins and impels us to define an "ideal protein" without inquiring whether the molecule actually occurs in nature.

Various salts employed as precipitant in combination with polyethylene glycol in protein/detergent particle association.

Salt/polyethylene glycol (PEG) mixtures are employed as precipitants for biological macromolecules. The dependence of precipitation curves (PCs) on salt species was investigated for integral membrane protein/detergent particles. By relating this dependence to properties of ions dissociated from added salts, the following roles and effects of various ions were clarified. In the presence of ions whose interaction with water is stronger than water-water interaction, the coordination of solvent molecules is rearranged so as to strengthen short-range steric repulsion and hydrophobic attraction. Ions whose interaction with water is weaker than water-water interaction can be a hindrance to hydrophobic-hydrophobic contact. Moreover, strong electric fields of divalent cations can cause an attractive effect between electronegative or polar groups of neighboring particles. The variations of particle-particle and particle-PEG interactions depending on the state of particles and surrounding solvents were correlative. Due to this, the relationship between the horizontal positions of PC and the species of salts added could be formulated as a binary linear function of cationic and anionic species composing the salts.

Data in support of intermolecular interactions at early stage of protein/detergent particle association induced by salt/polyethylene glycol mixtures.

The data provide information in support of the research article, "Intermolecular interactions at early stage of protein/detergent particle association induced by salt/polyethylene glycol mixtures" [1]. The data regarding variation of absorption spectra is used as an indicator of the duration of Rp. viridis PRU and RC, Rb. sphaeroides RC and LH2, and Rb. capsulatus LH2 in the native state in the presence of NaCl/polyethylene glycol (PEG) mixture. The data about minimum concentrations of salt and PEG whose aqueous phases are mutually separated presents information on additional influence of Tris buffer and N-octyl-ß-d-glucoside on the salt-PEG phase separation.

Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents.

As a first step toward the establishment of practical guidelines for the search for crystallization conditions, stability and solubility were examined for integral membrane proteins from photosynthetic bacteria in the presence of different detergents. The results obtained from their stability provided practical information on the proper choice of detergent type in the preparation process and the subsequent crystallization experiment. In addition, the determination of a solubility diagram provided a practical method for quantifying the correct choice of detergent concentration and for setting up the suitable precipitant concentration in the crystallization experiment.

Crystallization and preliminary X-ray studies on the reaction center-light-harvesting 1 core complex from Rhodopseudomonas viridis.

The reaction center-light-harvesting 1 (RC-LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (alpha, beta and gamma chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 A resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 A, alpha = 104.6, beta = 94.0, gamma = 110.7 degrees. A Patterson function calculated using data between 15.0 and 8.0 A resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.

Optimizing pH response of affinity between protein G and IgG Fc: how electrostatic modulations affect protein-protein interactions.

Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by "enthalpy-entropy compensation." Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.

Amyloid fibril formation by the CAD domain of caspase-activated DNase.

Caspase-activated DNase (CAD) is a key protein in the process of apoptosis that degrades DNA through the action of caspases. Its N-terminal region, the CAD domain (CAD-CD), is highly conserved among CAD family proteins and is responsible for the interaction with its inhibitor. We report here that CAD-CD spontaneously aggregates to form amyloid fibrils, without a lag time, under the conditions of low pH (below 4) and the presence of anions. Interestingly, the secondary structure of CAD-CD in the fibril state comprised not only beta-sheet but also alpha-helix, as found in CD, FTIR, and x-ray fiber diffraction experiments. Aromatic side chains have a defined orientation and are in the hydrophobic environment occurring with the CAD-CD fibrillogenesis. These findings provide new insights into the architecture of amyloid fibrils.

Structural genomics of membrane proteins.

A program on the structural genomics of membrane proteins has started at the BIRC, AIST, involving other academic institutions and industrial companies. Emphasis is being put on the development of techniques for the structural determination of membrane proteins of biological importance and ligand-receptor interactions by means of electron microscopy, X-ray diffraction, NMR, and computer simulation. Most efforts at the present stage, however, are being directed to finding suitable expression and purification systems and crystallization conditions for such proteins. The program is expected to be linked with the human full-length cDNA project and should lead to medical and industrial uses.