RESUMEN
We report an atomic force microscopy (AFM) study of fibrinogen molecules and fibrin fibers with resolution previously achieved only in few electron microscopy images. Not only are all objects triads, but the peripheral D regions are resolved into the two subdomains, apparently corresponding to the ßC and γC domains. The conformational analysis of a large population of fibrinogen molecules on mica revealed the two most energetically favorable conformations characterized by bending angles of â¼100 and 160 degrees. Computer modeling of the experimental images of fibrinogen molecules showed that the AFM patterns are in good agreement with the molecular dimensions and shapes detected by other methods. Imaging in different environments supports the expected hydration of the fibrinogen molecules in buffer, whereas imaging in humid air suggests the 2D spreading of fibrinogen on mica induced by an adsorbed water layer. Visualization of intact hydrated fibrin fibers showed cross-striations with an axial period of 24.0 ± 1.6 nm, in agreement with a pattern detected earlier with electron microscopy and small-angle X-ray diffraction. However, this order is clearly detected on the surface of thin fibers and becomes less discernible with the fiber's growth. This structural change is consistent with the proposal that thinner fibers are denser than thicker ones, that is, that the molecule packing decreases with the increasing of the fibers' diameter.
Asunto(s)
Fibrina/química , Fibrinógeno/química , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.
Asunto(s)
Fibrinógeno/química , Fibrinógeno/metabolismo , Microscopía de Fuerza Atómica , Adhesivos/química , Adhesivos/metabolismo , Células HEK293 , HumanosRESUMEN
Natural pore-forming proteins act as viral helical coats and transmembrane channels, exhibit antibacterial activity and are used in synthetic systems, such as for reversible encapsulation or stochastic sensing. These diverse functions are intimately linked to protein structure. The close link between protein structure and protein function makes the design of synthetic mimics a formidable challenge, given that structure formation needs to be carefully controlled on all hierarchy levels, in solution and in the bulk. In fact, with few exceptions, synthetic pore structures capable of assembling into periodically ordered assemblies that are stable in solution and in the solid state have not yet been realized. In the case of dendrimers, covalent and non-covalent coating and assembly of a range of different structures has only yielded closed columns. Here we describe a library of amphiphilic dendritic dipeptides that self-assemble in solution and in bulk through a complex recognition process into helical pores. We find that the molecular recognition and self-assembly process is sufficiently robust to tolerate a range of modifications to the amphiphile structure, while preliminary proton transport measurements establish that the pores are functional. We expect that this class of self-assembling dendrimers will allow the design of a variety of biologically inspired systems with functional properties arising from their porous structure.
Asunto(s)
Biopolímeros/química , Biopolímeros/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Transporte Biológico , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Modelos Moleculares , Porosidad , Estructura Cuaternaria de Proteína , Protones , EstereoisomerismoRESUMEN
We describe the self-assembly of multilayer hexagonal DNA arrays through highly regular interlayer packing. Slow cooling of a mixture of three single-stranded DNA sequences with various Mg2+ concentrations leads to the self-assembly of diverse multilayer architectures. The self-assembled aggregates were deposited onto mica surfaces and examined with atomic force microscopy. The size of the two-dimensional arrays and subsequent stacking to form multilayer structures are highly dependent on Mg2+ concentration. DNA bilayers and multilayers of defined shape are favored in 2-5 mM Mg2+ with an average lateral size of 700 nm. Arrays are much larger (up to 20 microm across) in 10-15 mM Mg2+, although multiple layers still make up 20-60% of the observed structures. Domains within single layer architectures were identified using Moiré pattern analysis. Distinct structural phases within the multilayer assemblies include two layers translated by 17.5 nm and interlayer rotations of 20 degrees and 30 degrees. Three layer assemblies have cubic close packing and taller multilayer architectures of 2D DNA sheets were also identified.
Asunto(s)
ADN/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Silicatos de Aluminio/química , Membrana Dobles de Lípidos/química , Magnesio/química , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Acid-base switchable supramolecular dendronized polyacetylenes (DPAs) with increasing steric bulk on going from generation one [G1] to three [G3], were constructed using multiple self-assembly processes between Fréchet-type [G1]-[G3]-dendritic dialkylammonium salts and a dibenzo[24]crown-8-containing polymer. The formation of the supramolecular systems is acid-base switchable to either an ON (rodlike dendronized polymers) or an OFF (flexible polymers) state. Thus, by controlling the superstructures of the supramolecular polymers with the [G1]-[G3] dendrons, it is possible to induce conformational changes within the polymer backbones. The supramolecular dendronized polymers, as well as their threading-dethreading properties, were characterized by (1)H NMR and UV absorption spectroscopies, gel permeation chromatography (GPC) and light scattering (LS). Independent measures of molecular weight (GPC, LS) indicate that DPAs behave as increasingly rigid macromolecules with each generation in solution. Molecular dynamics simulations of each DPA suggest that the lengths of the polymer backbones increase accordingly. Atomic force microscopy of the [G3]-dendronized polystyrene (DPS), as well as the DPAs, reveal surface morphologies indicative of aggregated superstructures.
RESUMEN
A library of eleven high cis-content cis-transoidal polyphenylacetylenes (PPAs) dendronized with self-assembling dendrons was prepared from a library of fifteen convergently synthesized macromonomers. Using [Rh(C triple bond CPh)(nbd)(PPh(3))(2)] (nbd=2,5-norbornadiene) in the presence of 10 equiv of N,N-dimethylaminopyridine, predictive control over molecular weight and narrow molecular weight distribution are obtained. The PPA backbone serves as a helical scaffold for the self-assembling dendrons. The dendron primary structure dictates the diameter of the cylindrical PPAs in bulk, both in the self-organized hexagonal columnar (Phi(h)) lattice determined by X-ray diffraction (XRD) and in monolayers on highly ordered pyrolytic graphite (HOPG) and mica visualized by atomic force microscopy (AFM). Thermal and bulk phase characteristics of the cylindrical PPAs reinforces the generality that flexible polymer backbones adopt a helical conformation within the cylindrical macromolecules generated by polymers jacketed with self-assembling dendrons.
RESUMEN
Oligo(N(6)-carbobenzyloxy-L-lysine) (OCBL) with n = 8 (n is the number-average degree of polymerization) was synthesized by the n-propylamine-catalyzed ring-opening polymerization of N(6)-carbobenzyloxy-L-lysine N-carboxylic anhydride, which was derived from N(6)-carbobenzyloxy-L-lysine. The formation of two-dimensionally well-ordered strip array monolayer films of the OBCL oligopeptide on graphite substrates was first succeeded by a conventional solution spin-coating process. The ordered strip array monolayer structure was characterized in detail by atomic force microscopy, and its assembly mechanism was examined.