Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

荧光蛋白报告基因在植物寄生真菌研究中的应用

综述菌根真菌、植物内生菌和植物病原真菌等植物寄生真菌转荧光蛋白基因研究现状.介绍转化载体的构建、转化方法及特基因的检测方法,以及转荧光蛋白基因技术在植物寄生真菌侵染过程研究中的应用,指出真菌转荧光蛋白基因存在的问题和展望.     

Variants of Green Fluorescent Protein GFPxm

As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could produce strong fluorescence when expressed at 37°C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37°C, and mutants GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42°C. Meanwhile, at least 4 mutants could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak.     

Investigation of the morphology of intermediate filaments adsorbed to different solid supports

Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with a width of 8-12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. In such conditions, human vimentin and desmin IFs appeared compact with a height of 5-8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had a height of only 3-5 nm, whereas desmin IFs appeared as 8-10 nm height filaments with a helical twist. Neurofilaments were 10-12 nm in height with a pronounced 30-50 nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example, broad, flattened filaments. Finally, hydrophilic glass was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed on this support. These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished from each others by their physicochemical properties.     

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.     

Visualizing association of lipidated signaling proteins in heterogeneous membranes−Partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association

In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 °C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered > liquid-ordered ? solid-ordered. Intriguingly, we detect - using the better spatial resolution of AFM - also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.     

Protein oligomerization through domain swapping: role of inter-molecular interactions and protein concentration

Domain swapping has been shown to be an important mechanism controlling multiprotein assembly and has been suggested recently as a possible mechanism underlying protein aggregation. Understanding oligomerization via domain swapping is therefore of theoretical and practical importance. By using a symmetrized structure-based (Gō) model, we demonstrate that in the free-energy landscape of domain swapping, a large free-energy barrier separates monomeric and domain-swapped dimeric configurations. We investigate the effect of finite monomer concentration, by implementing a new semi-analytical method, which involves computing the second virial coefficient, a thermodynamic indicator of inter-molecular interactions. This method, together with the symmetrized structure-based (Gō) model, minimizes the need for expensive many-protein simulations, providing a convenient framework to investigate concentration effect. Finally, we perform direct simulations of domain-swapped trimer formation, showing that this modeling approach can be used for higher-order oligomers.     

Protein content and amino acid composition of cassava leaf

On the basis of leaf dry wt, the protein content of six varieties of cassava varied from 29.3 to 38.6% and the estimated leaf protein production ranged from 242 to 953 kg per ha. On the basis of fr. wt of leaf, the total amino acids ranged from 8.42 to 9.4% while the essential amino acids averaged 4.21% and the sulphur-containing amino acids only 0.25%. The amino acid composition profiles for the six varieties was similar.     

Towards single-molecule DNA sequencing: assays with low nonspecific adsorption

New DNA sequencing techniques are currently being developed using single-molecule fluorescence-based detection of enzymatic double-strand synthesis. Such application requires surface architectures on which single-stranded templates can be immobilized. A further important attribute is a very low tendency to attract fluorescently labeled bases nonspecifically. On this account, the adsorption behaviour of Cy5-dNTPs on a variety of surface coatings was studied by performing real-time measurements of the DNA synthesis using a supercritical angle fluorescence biosensor. It is demonstrated that polyacrylic acid coatings are an excellent choice to minimize the nonspecific binding of the bases.     

Protein co-evolution, co-adaptation and interactions

Co-evolution has an important function in the evolution of species and it is clearly manifested in certain scenarios such as host–parasite and predator–prey interactions, symbiosis and mutualism. The extrapolation of the concepts and methodologies developed for the study of species co-evolution at the molecular level has prompted the development of a variety of computational methods able to predict protein interactions through the characteristics of co-evolution. Particularly successful have been those methods that predict interactions at the genomic level based on the detection of pairs of protein families with similar evolutionary histories (similarity of phylogenetic trees: mirrortree). Future advances in this field will require a better understanding of the molecular basis of the co-evolution of protein families. Thus, it will be important to decipher the molecular mechanisms underlying the similarity observed in phylogenetic trees of interacting proteins, distinguishing direct specific molecular interactions from other general functional constraints. In particular, it will be important to separate the effects of physical interactions within protein complexes (‘co-adaptation') from other forces that, in a less specific way, can also create general patterns of co-evolution.     

Binding and direct electrochemistry of OmcA, an outer-membrane cytochrome from an iron reducing bacterium, with oxide electrodes: A candidate biofuel cell system

Dissimilatory iron-reducing bacteria transfer electrons to solid ferric respiratory electron acceptors. Outer-membrane cytochromes expressed by these organisms are of interest in both microbial fuel cells and biofuel cells. We use optical waveguide lightmode spectroscopy (OWLS) to show that OmcA, an 85 kDa decaheme outer-membrane c-type cytochrome from Shewanella oneidensis MR-1, adsorbs to isostructural Al₂O₃ and Fe₂O₃ in similar amounts. Adsorption is ionic-strength and pH dependent (peak adsorption at pH 6.5-7.0). The thickness of the OmcA layer on Al₂O₃ at pH 7.0 [5.8 ± 1.1 (2σ) nm] from OWLS is similar, within error, to that observed using atomic force microscopy (4.8 ± 2 nm). The highest adsorption density observed was 334 ng cm⁻² (2.4 × 10¹² molecules cm⁻²), corresponding to a monolayer of 9.9 nm diameter spheres or submonolayer coverage by smaller molecules. Direct electrochemistry of OmcA on Fe₂O₃ electrodes was observed using cyclic voltammetry, with cathodic peak potentials of −380 to −320 mV versus Ag/AgCl. Variations in the cathodic peak positions are speculatively attributed to redox-linked conformation change or changes in molecular orientation. OmcA can exchange electrons with ITO electrodes at higher current densities than with Fe₂O₃. Overall, OmcA can bind to and exchange electrons with several oxides, and thus its utility in fuel cells is not restricted to Fe₂O₃.     

Probing macromolecular architectures of nanosized cyclic structures of (1-->3)-beta-D-glucans by AFM and SEC-MALLS

Comb-like branched (1-->3)-beta-D-glucans dissolve in water as stiff triple-helical structures. Dissociation followed by re-association leads to the formation of a blend of various macromolecular topologies, where the cyclic species make up a significant fraction. In this study, the molecular properties of these nanosized cyclic structures of (1-->3)-beta-D-glucans were probed using a combination of AFM and SEC-MALLS. The cyclic structures were obtained by subjecting linear triple-helical molecules of (1-->3)-beta-D-glucans to a denaturation-renaturation cycle, and the fraction of cyclic structures in the renatured sample was determined by AFM. Samples containing different known fractions of linear and circular molecules were studied by SEC with online multi-angle laser-light scattering and viscometric detectors. The molecular weight and the radius of gyration of the molecules eluting from the SEC column, as well as the concentration and the intrinsic viscosity, were determined simultaneously. By extrapolating the results to a situation of only circular species, the results allowed to determine the linear mass per unit length (M(L)) of not only the linear but also the circular morphologies of the (1-->3)-beta-D-glucans. The values obtained were M(L)=2140+/-180 g mol(-1)nm(-1) for the circular species and 2045+/-80 g mol(-1)nm(-1) for the linear species. This is the first direct determination of the M(L) parameter of the circular topology, and the results indicate that the reassociation of the individual chains yield a triplex structure also for the circular morphology, similar to the initial triple helix.     

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.     

Effects of interactions with micellar interfaces on the activity and structure of different lipolytic isoenzymes from Candida rugosa

The conformational and functional changes of cholesterol esterase (CE) and isolipase (CRL) from Candida rugosa after exposure to a micellar interface and subsequent extraction to a fresh buffer were studied. These two enzymes were activated by interaction with the micellar interface of a sulphosuccinic acid bis[2-ethylhexyl] ester/n-heptane/water system. For the hydrolysis of p-nitrophenyl butyrate ester in water, the catalytic efficiencies of CE and CRL were both improved because on activation their k_cat values increased from 378 to 465 and from 250 to 680 s⁻¹, respectively, while their K_m values decreased from 5.08 × 10⁻⁵ to 3.23 × 10⁻⁵ and from 2.28 × 10⁻⁴ to 1.14 × 10⁻⁴ M, respectively. After exposure to the micelles, CE showed a marked increase in its -helical content from 28 to 49%, but only limited changes were detected when CRL was exposed. These proteins exhibit similar capacities for increasing their -helical content in a helicogenic medium. In acetonitrile/water mixtures, CRL exhibits a partial decrease in the extent of its secondary structure, while CE exhibits an increase in its -helical content. The fact that this medium of reduced polarity permits one to simulate the effect of the AOT reverse micelles on the conformation of CE (increased helicity) but not their effect on the structure of CRL (decreased helicity) supports the hypothesis that only CE interacts to a significant extent with the apolar side of the micellar interface. After exposure to micelles of octyl-β-glucopyranoside, CE (but not CRL) showed a 10% increase in its -helical content.     

Protein location and elemental composition of urine spheres in different avian species

We examined the internal morphology, location of protein, and identity and location of elements, in avian urate-containing spheres in 9 species of birds. The urine spheres were collected from voided samples. The spheres ranged in size from 0.5-5.0 microm, except in the domestic fowl, where they ranged up to 10 microm in diameter. The internal morphology of the spheres was examined using freeze-fracture microscopy. Protein location within the spheres was identified using fluorescein isothiocyanate (FITC). The urine spheres were analyzed for content and internal location of elements using Energy Dispersal System Analysis (EDS). Internally, the spheres consisted of a central nidus surrounded by 3-4 concentric narrow rings of protein. Elements found within the spheres included nitrogen, potassium, calcium, sodium, phosphorus, chloride and sulfur; however, only nitrogen, potassium and chloride were common in the spheres of all species. Nitrogen comprised the majority of the elemental content of the spheres (77-90%) followed by potassium (8-45%), with all other ions present in trace amounts. Unlike protein, the location of elements was random within the spheres. Protein and urate are both negatively charged and known to associate to form the spheres and as potassium is the only cation common to all spheres, it too may play a role in their formation.     

The role of chromophore in the lipid-protein interactions in bacteriorhodopsin-phosphatidylcholine vesicles

By fluorescence and phase properties of a 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, the influence of the chromophore on the phase transition of bacteriorhodopsin–lipid vesicles was investigated. It was observed that removal of the chromophore led to the down-shifting of the phase transition temperatures. The temperatures corresponding to the beginning and ending of the gel–liquid phase transition were also influenced. This demonstrated that the liquid phase is reached more easily when the chromophore is bleached. The results indicate that removal of the chromophore alters the protein–lipid interactions. It is suggested that this alteration might be related to the change in the lipid molecular packing.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.