A solid-state NMR study of the dynamics and interactions of phenylalanine rings in a statherin fragment bound to hydroxyapatite crystals

Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, (13)C rotating frame relaxation measurements, as well as direct detection of correlations between (13)C spins on protein side chains and (31)P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide's N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.     

Adsorption behavior of statherin and a statherin peptide onto hydroxyapatite and silica surfaces by in situ ellipsometry

The salivary protein statherin is known to adsorb selectively onto hydroxyapatite (HA), which constitutes the main mineral of the tooth enamel. This adsorption is believed to be crucial for its function as an inhibitor of primary (spontaneous) and secondary (crystal growth) precipitation of calcium phosphate salts present in saliva. A fragment corresponding to the first 21 N-terminus amino acids of statherin (StN21) was previously found to reduce the rate of demineralization of HA. Therefore, the interfacial properties of this peptide and statherin onto silica, hydrophobized silica and HA discs was studied by in situ ellipsometry. Their reversibility induced by dilution and elutability induced by buffer and sodium dodecyl sulfate (SDS) was also determined. The results revealed that statherin adsorbed at a greater extent onto the HA as compared to StN21, suggesting that the hydrogen bonding between the uncharged polar residues at the C-terminal region of statherin and HA contributes to its adsorption. However, on both silica surfaces the peptide adsorption appeared to proceed in a similar way. Onto the hydrophobized silica the adsorption of both peptides was suggested to occur either via multilayer formation or adsorption of aggregates from solution, while onto the hydrophilic silica adsorption of peptide aggregates from solution was the suggested mechanism. Further, both peptides were observed to be strongly adsorbed onto HA, even after SDS treatment, in comparison to the layers adsorbed onto hydrophobized silica. Both peptide layers were found to be weakly adsorbed onto the hydrophilic silica surface as they were totally removed by buffer dilution.     

A REDOR NMR study of a phosphorylated statherin fragment bound to hydroxyapatite crystals

Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (15N) spin in the lysine side chain and two phosphorus-31 (31P) spins, at least one of which must be from the surface phosphates of the HAP.     

Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.     

Direct observation of phenylalanine orientations in statherin bound to hydroxyapatite surfaces

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.     

Conformational heterogeneity of surface-grafted amyloidogenic fragments of alpha-synuclein dimers detected by atomic force microscopy

A force-spectroscopy-based approach is used to characterize separation between amyloidogenic peptide fragments of alpha-synuclein. Interactions between individual molecules are studied using a scanning-force-microscopy-based technique. Alpha-synuclein fragments are attached to the solid surfaces via flexible long poly-(ethylene glycol) linkers removing aggregation state uncertainty of solution-based approaches and spurious surface effects. Tethering one fragment to the scanning probe tip and another fragment to the second surface ensures that interactions between tethered molecules are studied. Control experiments with only one tethered peptide indicate peptide-peptide interactions as the source of observed interaction forces in the double-tether experiment. The temperature dependence of rupture forces from 17.5 degrees C to 40 degrees C reveals similar molecular parameters indicating that no significant conformational changes occur in the associated molecules over this temperature range. Rate-dependent measurements indicate conformational heterogeneity of joined peptide molecules.     

Conformational changes of the amyloid beta-peptide (1-40) adsorbed on solid surfaces

The conformational change of the 39-43 residues of the amyloid beta-peptide (Abeta) toward a beta-sheet enriched state promotes self-aggregation of the peptide molecules and constitutes the major peptide component of the amyloid plaques in Alzheimer patients. The crucial question behind the self-aggregation of Abeta is related to the different pathways the peptide may take after cleavage from the amyloid precursor proteins at cellular membranes. This work is aiming at determining the conformation of the Abeta (1-40) adsorbed on hydrophobic Teflon and hydrophilic silica particles, as model sorbent surfaces mimicking the apolar transmembrane environment and the polar, charged membrane surface, respectively. The mechanism by which the Abeta interacts with solid surfaces strongly depends on the hydrophobic/hydrophilic character of the particles. Hydrophobic and electrostatic interactions contribute differently in each case, causing a completely different conformational change of the adsorbed molecules on the two surfaces. When hydrophobic interactions between the peptide and the sorbent prevail, the adsorbed Abeta (1-40) mainly adopts an alpha-helix conformation due to H-bonding in the apolar part of the peptide that is oriented towards the surface. On the other hand, when the peptide adsorbs by electrostatic interactions beta-sheet formation is promoted due to intermolecular association between the apolar parts of the adsorbed peptide. Irrespective of the characteristics of the solid sorbent, crowding the surface results in intermolecular association between adsorbed molecules leading to a strong aggregation tendency of the Abeta (1-40). [Diagram: see text] CD spectra of Abeta (1-40) at pH 7: A) in solution ([Abeta]=0.2 mg.ml(-1)) freshly prepared (line) and after overnight incubation (symbols);B) on Teflon (Gamma=0.5 mg.m(-2)).     

Synthesis of high surface area hydroxyapatite nanoparticles by mixed surfactant-mediated approach

A new surfactant-mediated approach was developed to synthesize hydroxyapatite (HAp) nanoparticles with high surface areas by calcination of their precursors encapsulated with calcium stearate using mixed surfactant-containing reaction mixtures. Acidic aqueous solution of calcium phosphate was mixed with both or either nonaoxyethylene dodecyl ether (C12EO9) and polyoxyethylene(20) sorbitan monostearate (Tween 60) and then was treated with aqueous ammonium at 25 degrees C. The C12EO9-based single surfactant system yielded an aggregate of platy HAp nanoparticles 20-40 nm in size, whereas the Tween 60-based single and mixed systems led to lath-shaped HAp nanoparticles 2-8 nm wide and encapsulated with calcium stearate. On calcination at 500 degrees C, the stearate-encapsulated HAp nanoparticles in the latter two systems were deorganized into high surface area HAp nanoparticles. Particularly, the HAp nanoparticles in the mixed system exhibited a specific surface area as high as 364 m2 g(-1) that is roughly 3 times larger than 160 m2 g(-1) for those in the single system. The significantly high surface area for the former is attributed to much less adhesion of decapsulated HAp nanoparticles, which originated from the particle-separating effect of the C12EO9 molecules adsorbed on the outer surface of the stearate-encapsulated HAp nanoparticles to inhibit their agglomeration or interfacial coordination. The present results demonstrate that the mixed use of two different surfactants as a source of encapsulating and templating agent and a particle-separating agent is specifically effective for the synthesis of high surface area HAp nanoparticles.     

A study on the melting and crystallization of polyoxymethylene‐copolymer/hydroxyapatite nanocomposites

In this work, isothermal crystallization kinetics of polyoxymethylene copolymer (POM) in POM/hydroxyapatite (HAp) nanocomposites has been investigated. Melting behavior and crystalline structure formation were studied using TOPEM DSC, positron lifetime spectroscopy (PALS), atomic force microscopy (AFM) and ¹³C and ³¹P solid‐state NMR. The highest degree of crystallinity was found for POM/0.5% HAp nanocomposite and the lowest for POM/2.5% HAp. Isothermal crystallization analysis showed that an introduction of HAp nanoparticles led to effective heterogeneous nucleation and formation of crystals with higher Avrami exponent. Besides, changes in overall crystallization rate were observed – the highest overall crystallization rate was found for POM/0.5% HAp sample, while the lowest for POM/2.5% HAp was observed. Generally, for POM in POM/HAp nanocomposites, a significant decrease in nucleation activation energy (K_g), and the fold surface free energy (σ_e) was found. For nanocomposite containing 2.5% HAp, heterogeneous nucleation takes place as well, but too high concentration of nanoparticles hinders POM crystallization and enhances formation of more defected crystals as confirmed by AFM data. The presence of HAp nanoparticles in the POM matrix was confirmed by ³¹P MAS‐NMR, but their influence on the crystallization process was not observed in the ¹³C CP‐MAS‐NMR spectra. Copyright © 2012 John Wiley & Sons, Ltd.     

羟基磷灰石负载Ni催化剂中Ni含量对催化甲烷二氧化碳重整制合成气性能的影响

以低温沉淀方法制备的羟基磷灰石(HAp)为载体,采用浸渍法制备了一系列不同Ni含量的Ni/HAp催化剂,并采用BET、H2-TPR、XRD、SEM、FT-IR、TEM和TG-DTA技术对催化剂进行了表征。结果表明,NiO含量为13%的催化剂表现出最好的催化甲烷二氧化碳重整制合成气活性,在850℃、空速3.6×104mL/(h·gcat)的反应条件下,甲烷和二氧化碳的转化率在10 h内分别稳定在72%和83%。这主要归因于催化剂中金属和载体之间的强相互作用。虽然反应后的催化剂表面有少量的积炭,但这些积炭多以丝状炭存在,并不会影响催化剂的活性和稳定性。     

Adsorption mechanism of polylysine on hydroxyapatite and its effect on dissolution properties of hydroxyapatite

Adsorbed amounts of poly-l-lysine (pLys) and bromide ion on hydroxyapatite (HAp) from aqueous solutions of poly-l-lysine hydrobromide, and amounts of calcium and phosphate ions liberated concurrently from HAp during the adsorption of pLys were determined at 25 degrees . The pLys was adsorbed on HAp by the mechanism of ion-exchange between its amino groups and calcium ions of HAp. The released amount of calcium ion increased, therefore, with the adsorbed amount of pLys. On the other hand, the released amount of phosphate ion first decreased and then increased after attaining a minimum with the equilibrium concentration of pLys. The analysis using an equilibrium dialysis method revealed that the released phosphate ions were mainly in the bound state to the amino groups of pLys remaining in the solution, and that the concentrations of calcium and phosphate ions free from both HAp and pLys were restricted by each other under the law of the solubility product of HAp. The first decrease in the released amount of phosphate ion was concluded to be attributed primarily to the increase in the released amount of calcium ion because pLys remaining in the solution was little in this region. When sodium hydroxide was added to the solution, the adsorbed amount of pLys increased and then slightly decreased with the equilibrium pH of the solution due to the increase or decrease of the electrostatic attractive force between the adsorbate and the adsorbent. However, conformational change in pLys around pH 10 seemed to have little effect on the adsorption.     

Temperature dependence of mucin adsorption

The kinetics of adsorption and desorption on a silica-like surface of the large glycoprotein mucin have been measured across a range of temperatures from 25 to 60 degrees C. The area occupied per molecule diminishes with increasing temperature both in the bulk and adsorbed states, implying that the glycoprotein belongs to the "natively open" conformational class. Due to the conformational rearrangement, the specific interaction energy governing desorption greatly increases with temperature, resulting in an impressively regulated temperature-invariant dynamic surface coating.     

DNA/羟基磷灰石杂化膜修饰电极用于研究DNA与量子点的相互作用

首次利用溶胶-凝胶一步法制备了纳米多孔羟基磷灰石(HAp)-DNA杂化膜修饰玻碳电极,HAp优良的生物相容性和独特的吸附性可以将DNA固定在HAp多孔薄膜上,而DNA的大分子结构对HAp膜起到稳定剂的作用.采用循环伏安法和交流阻抗法系统地研究了电极表面固定DNA的稳定性以及固定的DNA与非电活性核壳型量子点CdTe/C...     

Molecular simulation to characterize the adsorption behavior of a fibrinogen gamma-chain fragment

Implants invoke inflammatory responses from the body even if they are chemically inert and nontoxic. It has been shown that a crucial precedent event in the inflammatory process is the spontaneous adsorption of fibrinogen (Fg) on implant surfaces, which is typically followed by the presence of phagocytic cells. Interactions between the phagocyte integrin Mac-1 and two short sequences within the fibrinogen gamma chain, gamma190-202 and gamma377-395, may partially explain phagocyte accumulation at implant surfaces. These two sequences are believed to form an integrin binding site that is inaccessible when Fg is in its soluble-state structure but then becomes available for Mac-1 binding following adsorption, presumably due to adsorption-induced conformational changes. The objective of this research was to theoretically investigate this possibility by using molecular dynamics simulations of the gamma-chain fragment of Fg over self-assembled monolayer (SAM) surfaces presenting different types of surface chemistry. The GROMACS software package was used to carry out the molecular simulations in an explicit solvation environment over a 5 ns period of time. The adsorption of the gamma-chain of fibrinogen was simulated on five types of SAM surfaces. The simulations showed that this protein fragment exhibits distinctly different adsorption behavior on the different surface chemistries. Although the trajectory files showed that significant conformational changes did not occur in this protein fragment over the time frame of the simulations, it was predicted that the protein does undergo substantial rotational and translational motions over the surface prior to stabilizing in various preferred orientations. This suggests that the kinetics of surface-induced conformational changes in a protein's structure might be much slower than the kinetics of orientational changes, thus enabling the principles of adsorption thermodynamics to be used to guide adsorbing proteins into defined orientations on surfaces before large conformational changes can occur. This finding may be very important for biomaterial surface design as it suggests that surface chemistry can potentially be used to directly control the orientation of adsorbing proteins in a manner that either presents or hides specific bioactive sites contained within a protein's structure, thereby providing a mechanism to control cellular responses to the adsorbed protein layer.     

Simple method for the preparation of DNA-poly(o-methoxyaniline) hybrid: structure, morphology, and uncoiling of poly(o-methoxyaniline) on the DNA surface

The DNA-poly(o-methoxyaniline) (POMA) hybrid is prepared by mixing aqueous solutions of POMA [emeraldine salt (ES), doping level [Cl]/[N] = 0.52] and sodium salt of DNA (Na-DNA) and is then freeze-dried after 4 days. Three different compositions (WDNA = 0.25, 0.5, and 0.75, WDNA is the weight fraction of DNA) of the hybrids are prepared. The SEM pictures show a gradation in morphology; for example, for WDNA = 0.75, fibrils are present but, at lower DNA concentration, a fibrillar network structure of the hybrid is observed. The circular dichroism (CD) spectra of the hybrid solutions indicate unchanged DNA conformation and WAXS patterns indicate intact crystal structure of DNA in the hybrid. The UV-vis spectra suggest no denaturation of DNA during the blending process. The UV-vis spectra of the hybrids in aqueous medium show a gradual red shift of the pi band to polaron band transition with time. The plots of these wavelengths with time are sigmoidal, indicating the autocatalytic nature of the process. With an increase in the temperature, the rate of the red shift of the above peak is faster. Arrhenius analysis of the rate (1/tau700 where tau700 is the time required to reach the absorption maximum at the wavelength 700 nm) yields straight lines for the three hybrid compositions with activation energy values of 13-15 kcal/mol. These values are almost equal to the activation energy values of conformational transition of other polymers, supporting the red shift in UV-vis spectra occurs from conformational transition. DNA offers a surface where POMA gets adsorbed and the stable conformational transition resulting in the uncoiling of the POMA chain occurs through repulsive interaction among bound radical cations of POMA (ES) on the DNA surface. The conductivity of the hybrids is on the order of 10(-6) S/cm, and the I-V characteristic curves indicate the semiconducting nature of the hybrids.     

Structural control of peptide-coated gold nanoparticle assemblies by the conformational transition of surface peptides

Gold nanoparticles having peptide chains on the surfaces have been prepared yb ring-opening polymerization of gamma-methyl L-glutamate N-carboxyanhydride with fixed amino groups on the nanoparticle surface as an initiator. The number of peptide chains on the surface was adjusted to ca. 2 molecules per gold nanoparticle by controlling the number of fixed amino groups on the surface. The peptide chains on the surface were partially saponified to obtain poly(gamma-methyl L-glutamate-co-L-glutamic acid) with 28 mol% of glutamic acid residues. The number-average molecular weight of the peptide was 73,000. We described structural control of the peptide-coated gold nanoparticle assembly by conformational transition of the surface peptides. In deionized water, the peptide chains on the nanoparticle took a random coil conformation, and the individual nanoparticles existed in dispersed globular species. On the other hand, the peptide chains on the nanoparticle took an alpha-helical conformation in trifluoroethanol. Under this condition, the alpha-helical peptide chains on distinct gold nanoparticles connected the nanoparticles to form a fibril assembly owing to the dipole-dipole interaction between the surface peptide chains. The morphology of the peptide-coated gold nanoparticle assembly could be controlled by the conformational transition of surface peptides, which was attended by solution composition changes.     

Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores

The interactions of proteins with the surface of cylindrical nanopores are systematically investigated to elucidate how surface curvature and surface chemistry affect the conformation and activity of confined proteins in an aqueous, buffered environment. Two globular proteins, lysozyme and myoglobin, with different catalytic functions, were used as model proteins to analyze structural changes in proteins after adsorption on ordered mesoporous silica SBA-15 and propyl-functionalized SBA-15 (C(3)SBA-15) with carefully controlled pore size. Liquid phase ATR-FTIR spectroscopy was used to study the amide I and II bands of the adsorbed proteins. The amide I bands showed that the secondary structures of free and adsorbed protein molecules differ, and that the secondary structure of the adsorbed protein is influenced by the local geometry as well as by the surface chemistry of the nanopores. The conformation of the adsorbed proteins inside the nanopores of SBA-15 and C(3)SBA-15 is strongly correlated with the local geometry and the surface properties of the nanoporous materials, which results in different catalytic activities. Adsorption by electrostatic interaction of proteins in nanopores of an optimal size provides a favorably confining and protecting environment, which may lead to considerably enhanced structural stability and catalytic activity.     

应用顶空-气相离子迁移谱分析百合发酵乳的风味物质

按方法采集百合鳞茎经蒸制、打浆制成熟制样品(ZBH)。另取纯牛奶经杀菌、接种、发酵12h制得原味酸奶(SN-1/2)。另按方法制得百合酸奶(SN-3/4)和5个不同发酵时间(8,10,12,14,16h)的百合发酵酸奶(BHSN)。分别取上述4种样品各1.0g,分别按工作条件进行顶空采样并引入气相离子迁移谱分析系统进行分析。根据所测得数据,并经NIST数据库检索,在以上4种样品的挥发性组分中共鉴定了24种化合物。将这些化合物的信号峰强度进一步分析,可得到其中15种挥发物成分在不同样本中存在明显差异。根据所测得4种样品的挥发物中的一些特征组分或其信号峰强度的差异可对所涉及样品作出区别。例如正己醇等可作为ZBH的特征性组分。此外,对样品的风味物质进行正交偏最小二乘法判别分析(OPLS-DA),还可准确区分上述4种样品,以及不同发酵时间的BHSN的主要风味挥发成分的类别和含量,从而判断和选择适当的发酵时间。     

Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel

The structure of a water-insoluble fragment encompassing residues 282-304 of the HIV envelope protein gp41 is studied when solubilized by dihexanoyl phosphatidylcholine (DHPC) and by small bicelles, consisting of a 4:1 molar ratio of DHPC and dimyristoyl phosphatidylcholine (DMPC). Weak alignment with the magnetic field was accomplished in a stretched polyacrylamide gel, permitting measurement of one-bond (1)H-(15)N, (13)Ca-(13)C', and (13)C'-(15)N dipolar couplings, which formed the basis for determining the peptide structure. In both detergent systems, the peptide adopts an alpha-helical conformation from residue 4 through 18. In the presence of the DHPC micelles the helix is strongly curved towards the hydrophobic surface, whereas in the presence of bicelles a much weaker curvature in the opposite direction is observed.