Andrew N. Round

A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy

Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated number-average and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk.

Heterogeneity and Persistence Length in Human Ocular Mucins

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.

Unexpected branching in pectin observed by atomic force microscopy

Abstract Pectic polysaccharides extracted from unripe tomato plant cell walls have been imaged with an atomic force microscope (AFM). The images obtained reveal for the first time a branched structure for tomato pectins that differs from that proposed for the neutral sugar side chains from enzymatic hydrolysis and sugar analysis. The branches are between 30 and 170 nm long and are relatively linear. This work demonstrates that the AFM is uniquely capable of unambiguously identifying, with minimal sample preparation, individual macromolecules within a heterogeneous population.

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 cuticles 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 cutins elastic properties.

Lamellar structures of MUC2-rich mucin: a potential role in governing the barrier and lubricating functions of intestinal mucus.

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.

Exploring the consequences of attractive and repulsive interaction regimes in tapping mode atomic force microscopy of DNA

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.

Enhanced imaging of DNA via active quality factor control

Adsorption processes at single molecule level are of fundamental importance for the understanding and development of biomaterials. Atomic force microscopy (AFM) has played a critical role in this field due to its high resolution and ability to image in a liquid environment. We present a method that improves the dynamic force sensitivity and the resolution of a conventional AFM. This is achieved via a positive feedback loop that enhances the effective quality factor of the cantilever in a liquid environment to values in excess of 300, compared to a nominal value of ∼1. This active quality factor enhancement has been used to image DNA and an increase in the height of the molecule observed.

Characterising semi-refined iota-carrageenan networks by atomic force microscopy

Samples of PNG iota carrageenan have been studied by X-ray diffraction and Atomic Force Microscopy (AFM). Aqueous preparations of PNG iota carrageenan can be fractionated into water soluble and water insoluble components. The water soluble component is largely composed of iota carrageenan containing small amounts of cellulose. AFM studies reveal that this component forms network structures very similar to those obtained for refined iota carrageenan. Some swollen cellulosic networks were observed in the PNG samples. The water insoluble fraction has been shown to be predominately partially crystalline cellulose I. AFM reveals that the cellulose is present as a fibrous network similar to that found for bacterial cellulose or the cellulosic component of plant cell walls. In the PNG samples the lateral ordering of the cellulose microfibrils has been disrupted presumably during the extraction of the PNG carrageenan from the seaweed. The AFM imaging conditions used to study the insoluble component would not permit imaging of any iota carrageenan networks which may be present in this sample.

Mechanistic and Kinetic Insight into Spontaneous Cocrystallization of Isoniazid and Benzoic Acid

Solid-state cocrystallization is of contemporary interest because it offers an easy and efficient way to produce cocrystals, which are recognized as prospective pharmaceutical materials. Research explaining solid-state cocrystallization mechanisms is important but still too scarce to give a broad understanding of factors governing and limiting these reactions. Here we report an investigation of the mechanism and kinetics of isoniazid cocrystallization with benzoic acid. This reaction is spontaneous; however, its rate is greatly influenced by environmental conditions (humidity and temperature) and pretreatment (milling) of the sample. The acceleration of cocrystallization in the presence of moisture is demonstrated by kinetic studies at elevated humidity. The rate dependence on humidity stems from moisture facilitated rearrangements on the surface of isoniazid crystallites, which lead to cocrystallization in the presence of benzoic acid vapor. Furthermore, premilling the mixture of the cocrystal ingredients eliminated the induction time of the reaction and considerably increased its rate.

Supramolecular amino acid based hydrogels: probing the contribution of additive molecules using NMR spectroscopy

Abstract 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.