The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).

Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.     

Dehydration stability of amyloid fibrils studied by AFM

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Aβ(1–42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Aβ(1–42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.     

Gelation of chemically cross-linked methylcellulose studied by DSC and AFM

Gelation of methylcellulose (MC) and chemically cross-linked MC via urethane linkage (MCPU) with various molecular weights was investigated in a concentration range from 0.1 to 4.0 wt %. On heating of aqueous solution of MC, three transitions, clear sol to turbid sol, sol to gel and phase separation due to water separation were observed. With increasing molecular weight the transition temperatures decrease. In contrast, no effect of molecular weight on the transition temperatures was observed for MCPU. Structural change of water restrained by MC and MCPU was investigated by DSC. Melting and crystallization of water in both series of sample showed no significant difference, however, a molecular weight dependency of the glass transition was observed for MC. The results obtained in this study indicate that hydrophobic aggregation is restricted by cross-linking. Images of atomic force microscopy (AFM) indicated that from 6 to 16 molecules piled in two layers form a bundle. By chemical cross-linking, molecular chains align in the mono layer, molecular bundles consisting of more than 10 molecules coaggregate and form a large flexible ring.     

Hydration-dehydration of adsorbed protein films studied by AFM and QCM-D

The hydration-dehydration process of an adsorbed human serum albumin film has been studied using atomic force microscopy (AFM) and a quartz crystal microbalance (QCM). All measurements were performed with identically prepared protein films deposited on highly hydrophilic substrates. Both techniques are shown to be suitable for following in situ the kinetics of protein hydration, and for providing quantitative values of the adsorbed adlayer mass. The results obtained by the two methods have been compared and combined to study changes of physical properties of the films in terms of viscosity, shear, Young's modulus, density and film thickness. These properties were found to be reversible during hydration-dehydration cycles.     

Potato starch granule nanostructure studied by high resolution non-contact AFM

Surface studies at ambient conditions of potato starch granules subjected to multiple freezing and thawing, performed by a high resolution non-contact atomic force microscopy (nc-AFM), revealed some details of the starch granule nanostructure. After the treatment, a significant separation and a chain-like organisation of the granule surface elements have been observed. An accurate analysis of the granule surface nanostructure with a single amylopectine cluster resolution could be carried out. The oblong nodules of approximately 20-50 nm in diameter have been observed at the surface of the potato starch granules. The same size particles were precipitated by ethanol from gelatinized potato starch suspensions. They were also detected at the surface of oat and wheat starch granules. After multiple freezing and thawing, the eroded potato granule surface revealed a lamellar structure of its interior. The 30-40 nm inter-lamellar distances were estimated by means of nc-AFM. These findings fit previously proposed dimensions of the structural elements in the crystalline region of the starch granule. The observed surface sub-particles might correspond to the single amylopectine side chain clusters bundled into larger blocklets packed in the lamellae within the starch granule. The results supported the blocklet model of the starch granule structure.     

Assembly of alpha-synuclein fibrils in nanoscale studied by peptide truncation and AFM

α-Synuclein (α-Syn) fibrils are the major component of Lewy bodies that are closely associated with the pathogenesis of Parkinson’s disease, but the mechanism for the fibril assembly remains poorly understood. Here we report using a combination of peptide truncation and atomic force microscopy (AFM) to elucidate the self-assembly and morphology of the α-Syn fibrils. The results show that protease K significantly slims the fibrils from the mean height of ∼6.6 to ∼4.7 nm, whereas chaotropic denaturant urea completely breaks down the fibrils into small particles. The in situ enzymatic digestion also results in thinning of the fibrils, giving rise to some nicks on the fibrils. Moreover, N- or C-terminally truncated α-Syn fragments assemble into thinner filaments with the heights depending on the peptide lengths. A nine-residue peptide corresponding to the homologous GAV-motif sequence can form very thin (∼2.2 nm) but long (>1 μm) filaments. Thus, the central sequence of α-Syn forms a fibrillar core by cross-β-structure that is flanked by two flexible termini, and the orientation of the fibril growth is perpendicular to the β-sheet structures.     

天花粉蛋白对红细胞损伤作用的AFM研究

目的：利用原子力显微镜（atomic force microscopy,简称AFM）观察红细胞（red blood cells,简称RBC）与天花粉蛋白（trichosanthin,简称TCS）作用后形态上的变化以及细胞膜的损伤情况。方法：将1.2mg/ml的TCS溶液与红细胞的PBS缓冲溶液（pH7.4)按1：4的比例混合,在35℃温度下作用2h后,用原子力显微镜观察受损的红细胞,与正常红细胞进行对比。结果：（1）与正常红细胞相比,与TCS作用后的红细胞的高度明显降低,凹陷部分更加明显。（2）对红细胞上小范围扫描成像的结果显示,受损后的红细胞膜表面结构发生了变化,膜表面颗粒排列的特征依然存在,但颗粒之间开始产生连接。结论：TCS能损伤红细胞膜,改变细胞膜的微结构,引起红细胞的溶血作用。     

Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed.     

Multi-bead-and-spring model to interpret protein detachment studied by AFM force spectroscopy

This article deals with the detachment of molecules (fibrinogen) from a surface studied experimentally with an atomic force microscope. The detachment (or rupture) forces are measured as a function of the retraction velocity and exhibit a clear dependence on this parameter, even though the interaction between the molecules and the surface are nonspecific. To interpret these data, a mechanical multi-bead-and-spring model is developed. It consists of one to several parallel, "molecular" springs connected to an extra spring representing the cantilever that is moved at constant velocity. The free end of each molecular spring terminates with a particle that interacts with the surface through a Lennard-Jones potential. This Brownian dynamics model is used to analyze the experimental findings. In the framework of this model, it appears that the fibrinogen molecule must be ascribed a stiffness much smaller than that of the cantilever. In addition, several bonds between the molecule and the surface must be taken into account for the range of the molecule-surface interaction not to be unrealistically small. In future work, this model will be extended to more complex mechanisms such as the detachment of cells from a surface.     

Scale-independent roughness value of cell membranes studied by means of AFM technique

The roughness of cell membrane is a very interesting indicator of cell's health state. Atomic Force Microscopy allows us to investigate the roughness of cell membrane in great detail, but the obtained roughness value is scale-dependent, i.e. it strongly depends on measurement parameters, as scanning area and step size. The scale-dependence of the roughness value can be reduced by means of data filtration techniques, that are not standardized at nanometric scale, especially as far as biological data are concerned. In this work, a new method, based on the changes of values of some roughness parameter (root mean square roughness and skewness) as a function of filtration frequencies, has been implemented to optimize data filtering procedure in the calculation of cell membrane roughness. In this way, a root mean square roughness value independent of cell shape, membrane micro-irregularities and measurement parameters can be obtained. Moreover, different filtration frequencies selected with this method allow us to discriminate different surface regimes (nominal form, waviness and roughness) belonging to the raw cell profile, each one related to different features of the cell surface.     

The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.     

On the Adsorption of Human Acidic Proline-rich Proteins (PRP-1 and PRP-3) and Statherin at Solid/liquid Interfaces

The objective of the present study was to investigate the adsorption of PRP-1, PRP-3 and statherin to solid surfaces in terms of dependence on concentration, the presence of electrolyte and surface wettability. Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates of pure PRP-1, PRP-3 and statherin onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. The initial film build-up was fast and plateaus were reached within 10 min at all concentrations for both types of surfaces and all proteins. The observed adsorption and calculated diffusion rates of PRP-1, PRP-3 and statherin, respectively, indicated that the initial adsorption was mass transport controlled at low concentrations. At hydrophobic surfaces, isotherm shapes and adsorbed amounts were similar for PRP-1 and PRP-3, while statherin adsorbed to a higher extent. At hydrophilic surfaces only PRP-1 adsorbed substantially, while for PRP-3 and statherin adsorbed amounts were low. The presence of Ca 2+ ions in the phosphate buffer solution increased the adsorption of statherin and PRP-3 on hydrophobic surfaces, while PRP-1 was unaffected. On hydrophilic surfaces, all three proteins adsorbed in higher amounts in NaCl, compared to CaCl 2 at similar ionic strength. It is concluded that acidic PRPs (PRP-1 and PRP-3) and statherin readily form films on a variety of materials and solution conditions, showing that their functions may be fulfilled under a wide range of conditions.     

Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.     

Force profiles of protein pulling with or without cytoskeletal links studied by AFM

To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.     

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, α-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.     

Permeability of alkylamines across phosphatidylcholine vesicles as studied by 1H-NMR

The exchange rate and enthalpy and entropy of activation of the diffusion of the first five n-alkylamines across egg phosphatidylcholine vesicles has been measured by 1H-NMR spectroscopy employing the 1:2 Gd3+-EDTA complex as a relaxation reagent. The permeability determined from the exchange rate of the ethyl through the pentyl derivatives increased sequentially with increasing chain length from 7.10(-7) to 4.10(-4) cm/s, respectively, at 25 degrees C. The permeability of methylamine was similar to that of ethylamine (1.10(-6) cm/s at 25 degrees C) and exhibited a relatively smaller entropy increase. The enthalpy of activation for the transfer reaction was high for all amine derivatives (20 kcal/mol). The entropy of activation increased with increasing chain length. The results indicate that the rate of diffusion is dominated by the partition into the membrane. Methylamine, being the smallest molecule in this series, can probably diffuse also through vacancies formed by the internal motions of the lipid chains.     

Extraction and Purification,Structural Characterization and Biological Activity of a Polysaccharide,PRP-1, from Plumeria rubra

Polysaccharides derived from the flowers of Plumeria rubra (PRP) have shown a variety of beneficial effects on improving human health. However, the structural features and bioactivities of PRP remain unclear. A novel neutral polysaccharide (named PRP-1) with a molecular weight of 23 kDa was extracted and purified from the flowers of P. rubra. PRP-1 was consisted of arabinose, galactose, glucose, xylose and mannose, with a molar ratio of 1.49: 27.89: 50.24: 13.02: 7.36. The structural characterization based on the methylation and 1D/2D nuclear magnetic resonance analyses indicated that PRP-1 was composed of →4)-Glcp-(1→, →4,6)-Glcp-(1→, →4)-Galp-(1→, →2)-Galp-(1→, t-Gal(p), →4)-Manp-(1→, →4,6)-Manp-(1→, t-Man(p), →2)-Xylp-(1→, and t-Xyl(p). Scanning electron microscopy revealed that PRP-1 possess a compact three-dimensional curling network structure in the terms of morphology. PRP-1 exhibited anti-inflammatory activity, which have moderate inhibitory effects on TNF-α and IL-6 production in lipopolysaccharide (LPS)-induced RAW 264.7 cells. In addition, PRP-1 showed ABTS, OH radicals scavenging and the Fe²⁺ chelating effects in a concentration dependent manner. In α-glucosidase inhibition assay, PRP-1 did not exhibit inhibitory activity. Overall, these results provide a scientific basis for the utilization of the flowers of P. rubra as a potential functional food ingredient.     

Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by time-resolved AFM

The sensitivity of phospholipase A(2) (PLA(2)) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA(2) is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA(2) preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA(2) for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.