Nanomechanics of Pectin-Linked ß-Lactoglobulin Nanofibril Bundles.

Nanofibrils of ß-lactoglobulin can be assembled into bundles by site-specific noncovalent cross-linking with high-methoxyl pectin (Hettiarachchi et al. Soft Matter 2016, 12, 756). Here we characterized the nanomechanical properties of bundles using atomic force microscopy and force spectroscopy. Bundles had Gaussian cross sections and a mean height of 17.4 ± 1.4 nm. Persistence lengths were calculated using image analysis with the mean-squared end-to-end model. The relationship between the persistence length and the thickness had exponents of 1.69-2.30, which is consistent with previous reports for other fibril types. In force spectroscopy experiments, the bundles stretched in a qualitatively different manner to fibrils, and some of the force curves were consistent with peeling fibrils away from bundles. The flexibility of pectin-linked nanofibril bundles is likely to be tunable by modulating the stiffness and length of fibrils and the ratio of pectin to fibrils, giving rise to a wide range of structures and functionalities.

Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe-host interactions.

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique ß-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Structural changes in cell wall pectins during strawberry fruit development.

Strawberry (Fragaria × anannasa Duch.) is one of the most important soft fruit. Rapid loss of firmness occurs during the ripening process, resulting in a short shelf life and high economic losses. To get insight into the role of pectin matrix in the softening process, cell walls from strawberry fruit at two developmental stages, unripe-green and ripe-red, were extracted and sequentially fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material as well as the per fresh weight contents of the different fractions decreased in ripe fruit. The largest reduction was observed in the pectic fractions extracted with a chelating agent (trans-1,2- diaminocyclohexane-N,N,N'N'-tetraacetic acid, CDTA fraction) and those covalently bound to the wall (extracted with Na2CO3). Uronic acid content of these two fractions also decreased significantly during ripening, but the amount of soluble pectins extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruit. Fourier transform infrared spectroscopy of the different fractions showed that the degree of esterification decreased in CDTA pectins but increased in soluble fractions at ripen stage. The chromatographic analysis of pectin fractions by gel filtration revealed that CDTA, water and, mainly PAW polyuronides were depolymerised in ripe fruit. By contrast, the size of Na2CO3 pectins was not modified. The nanostructural characteristics of CDTA and Na2CO3 pectins were analysed by atomic force microscopy (AFM). Isolated pectic chains present in the CDTA fractions were significantly longer and more branched in samples from green fruit than those from red fruit. No differences in contour length were observed in Na2CO3 strands between samples of both stages. However, the percentage of branched chains decreased from 19.7% in unripe samples to 3.4% in ripe fruit. The number of pectin aggregates was higher in green fruit samples of both fractions. These results show that the nanostructural complexity of pectins present in CDTA and Na2CO3 fractions diminishes during fruit development, and this correlates with the solubilisation of pectins and the softening of the fruit.

Unravelling the nanostructure of strawberry fruit pectins by endo-polygalacturonase digestion and atomic force microscopy.

Pectins analysed by AFM are visualized as individual chains, branched or unbranched, and aggregates. To investigate the nature of these structures, sodium carbonate soluble pectins from strawberry fruits were digested with endo-polygalacturonase M2 from Aspergillus aculeatus and visualized by AFM. A gradual decrease in the length of chains was observed as result of the treatment, reaching a minimum LN value of 22nm. The branches were not visible after 2h of enzymatic incubation. The size of complexes also diminished significantly with the enzymatic digestion. A treatment to hydrolyse rhamnogalacturonan II borate diester bonds neither affected chains length or branching nor complex size but reduced the density of aggregates. These results suggest that chains are formed by a mixture of homogalacturonan and more complex molecules composed by a homogalacturonan unit linked to an endo-PG resistant unit. Homogalacturonan is a structural component of the complexes and rhamnogalacturonan II could be involved in their formation.

Trehalose-containing hydrocolloid edible films prepared in the presence of transglutaminase.

In this article, edible hydrocolloid films were prepared by using Citrus pectins and the protein phaseolin in the presence of microbial transglutaminase, an enzyme able to catalyze isopeptide bonds between endo-protein-reactive glutamine and lysine residues. For the first time, trehalose, a nonreducing homodisaccharide into which two glucose units are linked together by a α-1,1-glycosidic linkage, was used as a component of hydrocolloid films constituted of both proteins and carbohydrates. Our data have demonstrated that these films act as very effective barriers to gases, especially to CO2 . They also present a high antioxidant capability as measured by the 2,2-diphenyl-1-picrylhydrazyl scavenging assay. In addition, the films were characterized using Atomic Force Microscopy, a powerful tool used to evaluate film surface topography and roughness. The results of our experiments clearly indicate that the trehalose-containing films prepared both in the presence and absence of transglutaminase are composed of nanoparticles with a smooth surface, having similar roughness values (Rα). In conclusion, according to barrier and antioxidant properties and to their structure, it is possible to consider the trehalose-containing films as innovative bioplastics potentially able to protect different kinds of foods.

Recognition of galactan components of pectin by galectin-3.

It has been reported that modified forms of pectin possess anticancer activity. To account for this bioactivity, it has been proposed that fragments of pectin molecules can act by binding to and inhibiting the various roles of the mammalian protein galectin 3 (Gal3) in cancer progression and metastasis. Despite this clear molecular hypothesis and evidence for the bioactivity of modified pectin, the structural origins of the "bioactive fragments" of pectin molecules are currently ill defined. By using a combination of fluorescence microscopy, flow cytometry, and force spectroscopy, it has been possible to demonstrate, for the first time, specific binding of a pectin galactan to the recombinant form of human Gal3. Present studies suggest that bioactivity resides in the neutral sugar side chains of pectin polysaccharides and that these components could be isolated and modified to optimize bioactivity.