Efficient Multiple Domain Ligation for Proteins Using Asparaginyl Endopeptidase by Selection of Appropriate Ligation Sites Based on Steric Hindrance.

Three domain fragments of a multi-domain protein, ER-60, were ligated in two short linker regions using asparaginyl endopeptidase not involving denaturation. To identify appropriate ligation sites, by selecting several potential ligation sites with fewer mutations around two short linker regions, their ligation efficiencies and the functions of the ligated ER-60s were examined experimentally. To evaluate the dependence of ligation efficiencies on the ligation sites computationally, steric hinderances around the sites for the ligation were calculated through molecular dynamics simulations. Utilizing the steric hindrance, a site-dependent ligation potential index was introduced as reproducing the experimental ligation efficiency. Referring to this index, the reconstruction of ER-60 was succeeded by the ligation of the three domains for the first time. In addition, the new ligation potential index well-worked for application to other domain ligations. Therefore, the index may serve as a more time-effective tool for multi-site ligations.

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis.

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.

Oligomeric Structural Transition of HspB1 from Chinese Hamster.

HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.

Structural Studies of Overlapping Dinucleosomes in Solution.

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates.

Abnormal protein aggregation tends to result in the formation of ß-sheet rich amyloid fibrils, which are related to various kinds of amyloidoses and neurodegenerative diseases. The susceptibility to aggregation of protein molecules is dealt with by proteostasis in living systems, in which molecular chaperones play an important role. Recently, several secreted proteins have been examined as extracellular chaperones with a potency to suppress the formation of amyloid fibrils, although the whole picture that includes their inhibition mechanisms is not yet understood. In this study, we investigated the inhibitory effect of fibrinogen (Fg), one of the extracellular proteins identified as a potential member of the group of chaperones, on fibril formation. Insulin B chain was used as an amyloid formation model system because its prefibrillar intermediate species in the nucleation phase were well characterized. We revealed that Fg efficiently inhibited amyloid fibril formation via a direct interaction with the surface of the prefibrillar intermediates. Small-angle X-ray scattering experiments and a stoichiometry analysis suggested a structural model in which the surface of the rod-shaped prefibrillar intermediates is surrounded by Fg molecules. From such a specific manner of interactions, we propose that the role of Fg is to disturb fibril growth by confining the nuclei even when the nucleation occurs inside the prefibrillar intermediate. The structural property of the B-chain intermediates complexed with Fg would provide insights into the general principles of the functions of chaperones and other potential chaperone-like proteins involved in amyloid-related diseases.

Direct Evidence for the Effect of Glycerol on Protein Hydration and Thermal Structural Transition.

The mechanisms of protein stabilization by uncharged solutes, such as polyols and sugars, have been intensively studied with respect to the chemical thermodynamics of molecular crowding. In particular, many experimental and theoretical studies have been conducted to explain the mechanism of the protective action on protein structures by glycerol through the relationship between hydration and glycerol solvation on protein surfaces. We used wide-angle x-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation to quantitatively characterize the hydration and/or solvation shell of myoglobin in aqueous solutions of up to 75% v/v glycerol. At glycerol concentrations below ∼40% v/v, the preservation of the hydration shell was dominant, which was reasonably explained by the preferential exclusion of glycerol from the protein surface (preferential hydration). In contrast, at concentrations above 50% v/v, the partial penetration or replacement of glycerol into or with hydration-shell water (neutral solvation by glycerol) was gradually promoted. WAXS results quantitatively demonstrated the neutral solvation, in which the replacement of hydrated water by glycerol was proportional to the volume fraction of glycerol in the solvent multiplied by an exchange rate (ß ≤ 1). These phenomena were confirmed by small-angle neutron scattering measurements. The observed WAXS data covered the entire hierarchical structure of myoglobin, ranging from tertiary to secondary structures. We separately analyzed the effect of glycerol on the thermal stability of myoglobin at each hierarchical structural level. The thermal transition midpoint temperature at each hierarchical structural level was raised depending on the glycerol concentration, with enhanced transition cooperativeness between different hierarchical structural levels. The onset temperature of the helix-to-cross ß-sheet transition (the initial process of amyloid formation) was evidently elevated. However, oligomerization connected to fibril formation was suppressed, even at a low glycerol concentration.

Elucidating the Solvent Effect on the Switch of the Helicity of Poly(quinoxaline-2,3-diyl)s: A Conformational Analysis by Small-Angle Neutron Scattering.

Small-angle neutron scattering (SANS) was used to examine dilute solutions of a poly(quinoxaline-2,3-diyl) (PQX) with (R)-2-octyloxymethyl side chains in deuterated THF or a mixture of deuterated 1,1,2-TCE and THF (8/2, v/v), in which the PQX adopts pure P- and M-helical structures. The structures of the PQX that were obtained based on the SANS experiments in combination with theoretical calculations suggest that in THF, the chiral side chains of the P-helical PQX are extended, whereas in the 1,1,2-TCE/THF mixture, the side chains of the M-helical PQX are folded. Consequently, P-helical structures should be preferred in good solvents such as THF, which solvate the extended side chains, whereas M-helical structures should be preferred in poor solvents such as 1,1,2-TCE, wherein the side chains adopt shrunken conformations with maximized van der Waals interactions between the side chains. This study thus reveals the first example for fully determined nuanced conformations of the side chains of synthetic polymers in solution based on SANS experiments and theoretical calculations.

Solution scattering approaches to dynamical ordering in biomolecular systems.

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Preparation, characterization and magnetic behavior of a spin-labelled physical hydrogel containing a chiral cyclic nitroxide radical unit fixed inside the gelator molecule.

An optically active amphiphilic nitroxide radical compound [(S,S,R)-], which contains a paramagnetic (2S,5S)-2,5-dimethyl-2,5-diphenylpyrrolidine-N-oxyl radical group fixed in the inner position together with a hydrophobic long alkyl chain and a hydrophilic (R)-alanine residue in the opposite terminal positions, was found to serve as a low-molecular-weight gelator in H2O to give rise to a spin-labelled physical hydrogel. Characterization of the hydrogel was performed by microscopic (SEM, TEM and AFM) techniques, XRD and SAXS measurements, and IR, UV and CD spectroscopies. The gel-sol transition temperature was determined by EPR spectral line-width (ΔHpp) analysis. Measurement of the temperature dependence of relative paramagnetic susceptibility (χrel) for the hydrogel and sol phases was achieved by means of the double-integration of VT-EPR spectra.

Distinct features of the histone core structure in nucleosomes containing the histone H2A.B variant.

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.

Selective Hg(II) adsorption from aqueous solutions of Hg(II) and Pb(II) by hydrolyzed acrylamide-grafted PET films.

Selective Hg(II) adsorption from aqueous solutions of Hg(II) and Pb(II) using hydrolyzed acrylamide (AAm)-grafted polyethylene terephthalate (PET) films was examined to explore the potential reuse of waste PET materials. Selective recovery of Hg(II) from a mixture of soft acids with similar structure, such as Hg(II) and Pb(II), is important to allow the reuse of recovered Hg(II). An adsorbent for selective Hg(II) adsorption was prepared by γ-ray-induced grafting of AAm onto PET films followed by partial hydrolysis through KOH treatment. The adsorption capacity of the AAm-grafted PET films for Hg(II) ions increased from 15 to 70 mg/g after partial hydrolysis because of the reduction of hydrogen bonding between -CONH2 groups and the corresponding improved access of metal ions to the amide groups. The prepared adsorbent was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The absorbent film showed high selectivity for the adsorption of Hg(II) over Pb(II) throughout the entire initial metal concentration range (100-500 mg/L) and pH range (2.2-5.6) studied. The high selectivity is attributed to the ability of Hg(II) ions to form covalent bonds with the amide groups. The calculated selectivity coefficient for the adsorbent binding Hg(II) over Pb(II) was 19.2 at pH 4.5 with an initial metal concentration of 100 mg/L. Selective Hg(II) adsorption equilibrium data followed the Langmuir model and kinetic data were well fitted by a pseudo-second-order equation. The adsorbed Hg(II) and Pb(II) ions were effectively desorbed from the adsorbent film by acid treatment, and the regenerated film showed no marked loss of adsorption capacity upon reuse for selective Hg(II) adsorption.

Determination of the point resolution of high-resolution transmission electron microscope using the through-focus technique.

From the viewpoint of evaluating the instrumental performance of high-resolution electron microscopy (HREM), the Scherzer condition was investigated using information theory. As a result, the optimum defocus amount Δf can be expressed based on [Formula: see text] , and the formula [Formula: see text] is obtained. Furthermore, a procedure for measuring point resolution using the through-focus technique is developed, and a new method for determining the spherical aberration coefficient using the variance of Δf is introduced in the procedure.

Characterization of ßB2-crystallin tryptophan mutants reveals two different folding states in solution.

Conserved tryptophan residues are critical for the structure and the stability of ß/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in ß/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of ß/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human ßB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of ßB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of ßB2-crystallin vary over time. By using SAXS, we found that the dimer of ßB2-crystallin in solution resembled the lattice ßB1-crystallin dimer (face-en-face), whereas the tetramer of ßB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of ßB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of ßB2-crystallin in lens.

Genome-wide mapping and cryo-EM structural analyses of the overlapping tri-nucleosome composed of hexasome-hexasome-octasome moieties.

The nucleosome is a fundamental unit of chromatin in which about 150 base pairs of DNA are wrapped around a histone octamer. The overlapping di-nucleosome has been proposed as a product of chromatin remodeling around the transcription start site, and previously found as a chromatin unit, in which about 250 base pairs of DNA continuously bind to the histone core composed of a hexamer and an octamer. In the present study, our genome-wide analysis of human cells suggests another higher nucleosome stacking structure, the overlapping tri-nucleosome, which wraps about 300-350 base-pairs of DNA in the region downstream of certain transcription start sites of actively transcribed genes. We determine the cryo-electron microscopy (cryo-EM) structure of the overlapping tri-nucleosome, in which three subnucleosome moieties, hexasome, hexasome, and octasome, are associated by short connecting DNA segments. Small angle X-ray scattering and coarse-grained molecular dynamics simulation analyses reveal that the cryo-EM structure of the overlapping tri-nucleosome may reflect its structure in solution. Our findings suggest that nucleosome stacking structures composed of hexasome and octasome moieties may be formed by nucleosome remodeling factors around transcription start sites for gene regulation.

Mechanistic Modeling of Amyloid Oligomer and Protofibril Formation in Bovine Insulin.

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for understanding pathogenesis. However, the detailed mechanisms of their formation have been difficult to elucidate because they tend to form transiently and heterogeneously. Here, we found that bovine insulin protofibril formation proceeds in a monodisperse manner, which allowed us to characterize the detailed early aggregation process by light scattering in combination with thioflavin T fluorescence and Fourier transform infrared spectroscopy. The protofibril formation was specific to bovine insulin, whereas no significant aggregation was observed in human insulin. The kinetic analysis combining static and dynamic light scattering data revealed that the protofibril formation process in bovine insulin can be divided into two steps based on fractal dimension. When modeling the experimental data based on Smoluchowski aggregation kinetics, an aggregation scheme consisting of initial fractal aggregation forming spherical oligomers and their subsequent end-to-end association forming protofibrils was clarified. Furthermore, the analysis of temperature and salt concentration dependencies showed that the end-to-end association is the rate-limiting step, involving dehydration. The established model for protofibril formation, wherein oligomers are incorporated as a precursor, provides insight into the molecular mechanism by which protein molecules assemble during the early stage of amyloid formation.

Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form.

A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and ß subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four ß subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

Change of dynamics of raft-model membrane induced by amyloid-ß protein binding.

While the steady-state existence in the size and shape of liquid-ordered microdomains in cell membranes, the so-called "lipid rafts", still remain the subject of debate, glycosphingolipid-cholesterol rich regions in plasma membranes have been considered to have a function as platforms for signaling and sorting. In addition, recent spectroscopic studies show that the interaction between monosialoganglioside and amyloid beta (Aß protein promotes the transition of Aß from the native structure to the cross-beta fold in amyloid aggregates. However, there is few evidence on the dynamics of "lipid rafts" membranes. As the neutron spin-echo (NSE) technique is well known to detect directly slow dynamics of membrane systems in situ, by the combination of NSE and small-angle X-ray scattering we have studied the effect of the interaction between raft-model membrane and amyloid Aß proteins on the structure and dynamics of a large uni-lamellar vesicle (LUV) consisting of monosialoganglioside-cholesterol-phospholipid ternary mixtures as a model of lipid-raft membrane. We have found that the interaction between the Aß proteins and the model membrane at the liquid crystal phase significantly suppresses a bending-diffusion motion with a minor effect on the LUV structure. The present results would suggest a possibility of non-receptor-mediated disorder in signaling through a modulation of a membrane dynamics induced by the association of amyloidogenic peptides on a plasma membrane.