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Atomic force microscopy (AFM) has been used to investigate the heterogeneity and flexibility of human ocular mucins and their subunits. We have paid particular attention, in terms of theory and experiment, to the problem of inducing the polymers to assume equilibrium conformations at a surface. Mucins deposited from a buffer containing Ni(2+) ions adopt extended conformations on mica akin to those observed for DNA under similar conditions. The heterogeneity of the intracellular native mucins is evident from a histogram of contour lengths, reflecting, in part, the diversity of mucin gene products expressed. Reduction of the native mucin with dithiothreitol, thereby breaking the S==S bonds between cysteine residues, causes a marked reduction in polymer length. These results reflect the modes of transport and assembly of newly synthesized mucins in vivo. By modifying the worm-like chain model for applicability to two dimensions, we have confirmed that under the conditions employed mucin adsorbs to mica in an equilibrated conformation. The determined persistence length of the native mucin, 36 nm, is consistent with that of an extended, flexible polymer; such characteristics will influence the properties of the gels formed in vivo.

Mucus is a ubiquitous feature of mammalian wet epithelial surfaces, where it lubricates and forms a selective barrier that excludes a range of particulates, including pathogens, while hosting a diverse commensal microflora. The major polymeric component of mucus is mucin, a large glycoprotein formed by several MUC gene products, with MUC2 expression dominating intestinal mucus. A satisfactory answer to the question of how these molecules build a dynamic structure capable of playing such a complex role has yet to be found, as recent reports of distinct layers of chemically identical mucin in the colon and anomalously rapid transport of nanoparticles through mucus have emphasized. Here we use atomic force microscopy (AFM) to image a MUC2-rich mucus fraction isolated from pig jejunum. In the freshly isolated mucin fraction, we find direct evidence for trigonally linked structures, and their assembly into lamellar networks with a distribution of pore sizes from 20 to 200 nm. The networks are two-dimensional, with little interaction between lamellae. The existence of persistent cross-links between individual mucin polypeptides is consistent with a non-self-interacting lamellar model for intestinal mucus structure, rather than a physically entangled polymer network. We only observe collapsed entangled structures in purified mucin that has been stored in nonphysiological conditions. ■ INTRODUCTIONMucus forms a protective and selective barrier as well as a lubricating film over wet epithelial surfaces in the mammalian body, including those of the respiratory, ocular, reproductive, and gastrointestinal (GI) systems.1,2 It consists of large glycoproteins (mucins) forming a viscoelastic gel. While many details of the structure of individual mucin polymers are well understood, 3 and many observations of the micro and macroscale rheology of mucus have been made, 4−10 it is apparent that there currently remain significant gaps in our understanding of the way in which the secreted mucin polymers are arranged so as to give rise to their observed behavior.11 A clearer understanding of this link will critically inform our understanding of how the mucus barrier works: how commensal and pathogenic bacteria interact with the mucosal environment, how drug delivery across the mucus barrier may be affected, and how physiological processes such as nutrient absorption after digestion take place. In particular, the prevailing view of GI mucins forming a shear-thinning, physically entangled gel is not a convincing model that allows mucus in the GI tract to act both as a barrier and as a lubricating layer if there are extensive cross-links between the mucins. Recent evidence suggests that protease-resistant trimeric cross-links are formed at the N-termini of MUC2 mucins, 12 the dominant mucin gene product in the small and large intestine, and so the model of GI mucin needs to be revisited if we are to form a clear picture of its function as a barrier and lubricant.Recently, several new findings have highlighted this gap in our und...

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.

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Supramolecular hydrogels are composed of self‐assembled solid networks that restrict the flow of water. l‐Phenylalanine is the smallest molecule reported to date to form gel networks in water, and it is of particular interest due to its crystalline gel state. Single and multi‐component hydrogels of l‐phenylalanine are used herein as model materials to develop an NMR‐based analytical approach to gain insight into the mechanisms of supramolecular gelation. Structure and composition of the gel fibres were probed using PXRD, solid‐state NMR experiments and microscopic techniques. Solution‐state NMR studies probed the properties of free gelator molecules in an equilibrium with bound molecules. The dynamics of exchange at the gel/solution interfaces was investigated further using high‐resolution magic angle spinning (HR‐MAS) and saturation transfer difference (STD) NMR experiments. This approach allowed the identification of which additive molecules contributed in modifying the material properties.

Tapping mode atomic force microscopy (TM-AFM) in an ambient environment is a widely employed tool in the field of characterization of materials at the nanoscale. Significant advances have recently been made in the understanding of the physics behind some of the complexities of its operation, the most profound being the prediction and demonstration of the existence of the attractive and repulsive regimes of tip–sample interaction. In this paper we present an investigation of the criteria required for accessing the two imaging regimes, a simple method for controlling the transition between them in situ, and an assessment of their consequences for topographic and phase shift images of DNA. We find that the transition from repulsive to attractive regime imaging is characterized by a large increase in topographic height and concomitant decrease and sign inversion of the phase shift recorded over single molecules of DNA on mica. By varying the frequency at which the cantilever is driven, we can select which regime we wish to operate in routinely and reproducibly. Controlling the tip–sample interaction in this way greatly improves images of fragile nanoscale structures such as single molecules.