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.

Characterization of Localization, Ligand Binding, and pH-Dependent Conformational Changes of Two Chemosensory Proteins Expressed in the Antennae of the Japanese Carpenter Ant, Camponotus Japonicus.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 Å increase) at pH 4.0 than at pH 7.0. The dissociation constant (Kd) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Short-Distance Intermolecular Correlations of Mono- and Disaccharides in Condensed Solutions: Bulky Character of Trehalose.

Organisms with tolerance to extreme environmental conditions (cryptobiosis) such as desiccation and freezing are known to accumulate stress proteins and/or sugars. Trehalose, a disaccharide, has received considerable attention in the context of cryptobiosis. It has already been shown to have the highest glass-transition temperature and different hydration properties from other mono- and disaccharides. In spite of the importance of understanding cryptobiosis by experimentally clarifying sugar-sugar interactions such as the clustering in concentrated sugar solutions, there is little direct experimental evidence of sugar solution structures formed by intermolecular interactions and/or correlation. Using a wide-angle X-ray scattering method with the real-space resolution from ∼3 to 120 Å, we clarified the characteristics of the structures of sugar solutions (glucose, fructose, mannose, sucrose, and trehalose), over a wide concentration range of 0.05-0.65 g/mL. At low concentrations, the second virial coefficients obtained indicated the repulsive intermolecular interactions for all sugars and also the differences among them depending on the type of sugar. In spite of the presence of such repulsive force, a short-range intermolecular correlation was found to appear at high concentrations for every sugar. The concentration dependence of the observed scattering data and p(r) functions clearly showed that trehalose prefers a more disordered arrangement in solution compared to other sugars, that is, bulky arrangement. The present findings will afford a new insight into the molecular mechanism of the protective functions of the sugars relevant to cryptobiosis, particularly that of trehalose.

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular-Crowding Environment of Cells Using Neutron Scattering and Cell Debris.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation have provided new insights into the structure and dynamics of biological molecules inside the cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background scattering from large amounts of cell debris, we successfully extracted structure information for both small globular protein and small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1:60 for protein/crowder and 1:40 for SUV/crowder.

Structure of Ultrafine Bubbles and Their Effects on Protein and Lipid Membrane Structures Studied by Small- and Wide-Angle X-ray Scattering.

Ultrafine bubbles (UFBs) are defined as small gas-filled bubbles with a diameter smaller than 1 µm. UFBs are stable for several weeks in aqueous solutions due to their small size. Although the mechanism of the stability of UFBs remains under intensive investigation, industrial applications of UFBs have recently arisen in various fields such as agricultural and fishery industries and medical therapy. The relevance of ions (protons and hydroxide anions) in UFB solutions has been discussed; however, the mechanism underlying the behavior of UFBs is still ambiguous and there is little direct evidence of the effect of UFBs on biological materials. This study deals with gaseous UFBs in aqueous solutions. Using small- and wide-angle X-ray scattering, we have investigated the structures of UFBs (air-UFBs, O2-UFBs, and N2-UFBs) and their effect on protein and lipid membrane structures. X-ray scattering and modeling data suggest that UFBs present a dynamic diffusive boundary (interface) due to the continuous release and absorption of gas. UFBs were found to not affect the structures of proteins at all hierarchal structure levels (from quaternary to tertiary, to internal, to secondary), whereas they did influence the packing and fluctuation of the hydrocarbon chains in the liposomes but not their shapes.

Restoration of Myoglobin Native Fold from Its Initial State of Amyloid Formation by Trehalose.

Organisms having tolerances against extreme environments produce and accumulate stress proteins and/or sugars in cells against the extreme environment such as high or low temperature, drying, and so forth. Sugars and/or polyols are known to prevent protein denaturation and enzyme deactivation. In particular, trehalose has received considerable attention because of its association with cryptobiosis and anhydrobiosis. This study focuses on the restoration of acid-denatured amyloid transition of myoglobin by trehalose. Myoglobin is known to proceed amyloidogenic reaction under denaturation conditions. We found that acid-denatured myoglobin at an initial process of amyloidogenic reaction (helix-to-sheet transition followed by oligomerization) at 25 °C was substantially restored to its native structure by trehalose. This action was prominent during the early stage of amyloid formation. Recent results showed that sugars are preferentially excluded from the protein surface to preserve its hydration shell and stabilize the protein structure against chemical and thermal denaturation. Therefore, the present results suggest that trehalose will restore the tightly bound water molecules around the hotspot (G-helix) of myoglobin on the amyloid transition by its intrinsic preservative action of the native hydration shell against denaturation. The present finding on the restorative action by trehalose could provide new insights into protein folding and amyloidosis.

Preferential Intercalation of Human Amyloid-ß Peptide into Interbilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment.

This study focuses on the interaction of human amyloid ß-peptide (Aß) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aß is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aß-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aß. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aß under noncrowding conditions. However, the addition of Aß under crowding conditions induced shape deformation and aggregation to SUV resulting in multilamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aß peptide into the interbilayer region. This phenomenon was only observed at the gel (Lß) phase. These results suggest that an intracellular crowding environment promotes Aß-membrane interaction and a selective accumulation of Aß peptides into the interbilayer regions.

Sugar-Mediated Stabilization of Protein against Chemical or Thermal Denaturation.

The protective action of sugars against the denaturation of myoglobin was clarified by X-ray and neutron scattering methods. Different types of sugars such as disaccharides (trehalose, sucrose) and monosaccharides (glucose, fructose) were used. Experimental data and theoretical simulation based on three different solvation models (preferential solvation model, nonpreferential solvation model, and preferential exclusion (hydration) model) indicated that sugar molecules were preferentially or weakly excluded from the protein surface and preserved the native protein hydration shell. This trend was more evident for disaccharides. The preferential exclusion shifted gradually to the nonpreferential solvation at higher sugar concentrations. On the protective actions of the sugars against the guanidinium-chloride-mediated denaturation, all sugars, starting from the low concentration of 5% w/v, showed the protective trend toward the protein native structure, especially for the secondary structure. The thermal structural transition temperature of myoglobin was raised by about 4-5 °C, accompanied by amyloid formation, for all hierarchical structural levels. In particular, the oligomer formation of the amyloid aggregates was more suppressed. The above protective action was sugar-dependent. The present results clearly suggest that sugars intrinsically protect the native structure of proteins against chemical and thermal denaturation through the preservative action of the hydration shell.

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.