Sliding Contact Dynamic Force Spectroscopy Method for Interrogating Slowly Forming Polymer Cross-Links

Dynamic Single Molecule Force Spectroscopy (SMFS), conducted most commonly using AFM, has become a widespread and valuable tool for understanding the kinetics and thermodynamics of fundamental molecular processes such as ligand-receptor interactions and protein unfolding. Where slowly forming bonds are responsible for the primary characteristics of a material, as is the case in crosslinks in some polymer gels, care must be taken to ensure that a fully equilibrated bond has first formed before its rupture can be interpreted. Here we introduce a method, sliding contact force spectroscopy (SCFS), which effectively eliminates the kinetics of bond formation from the measurement of bond rupture. In addition it permits bond rupture measurements in systems where one of the binding partners may be introduced into solution prior to binding without tethering to a surface. Taking as an exemplar of a slowly forming bond the ‘eggbox’ junction crosslinks between oligoguluronic acid chains (oligoGs) in the commercially important polysaccharide alginate, we show that SCFS measures accurately the equilibrated bond strength of the crosslink when one chain is introduced into the sample solution without tethering to a surface. The results validate the SCFS technique for performing single molecule force spectroscopy experiments, and show that it has advantages in cases where the bond to be studied forms slowly and where tethering of one of the binding partners is impractical

Strain Construction and Process Development for Efficient Recombinant Production of Mannuronan C-5 Epimerases in Hansenula polymorpha

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates.publishedVersio

NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been 13C, 15N isotopically labelled and recombinantly expressed. We report here the 1H, 13C, 15N resonance assignment of AlgE6R3

Structure–activity relationships of low molecular weight alginate oligosaccharide therapy against Pseudomonas aeruginosa

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)–DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures

Polymer sequencing by molecular machines: A framework for predicting the resolving power of a sliding contact force spectroscopy sequencing method

We evaluate an AFM-based single molecule force spectroscopy method for mapping sequences in otherwise difficult to sequence heteropolymers, including glycosylated proteins and glycans. The sliding contact force spectroscopy (SCFS) method exploits a sliding contact made between a nanopore threaded over a polymer axle and an AFM probe. We find that for sliding α- and β- cyclodextrin nanopores over a wide range of hydrophilic monomers, the free energy of sliding is proportional to the sum of two dimensionless, easily calculable parameters representing the relative partitioning of the monomer inside the nanopore or in the aqueous phase, and the friction arising from sliding the nanopore over the monomer. Using this relationship we calculate sliding energies for nucleic acids, amino acids, glycan and synthetic monomers and predict on the basis of these calculations that SCFS will detect N- and O-glycosylation of proteins and patterns of sidechains in glycans. For these applications, SCFS offers an alternative to sequence mapping by mass spectrometry or newly-emerging nanopore technologies that may be easily implemented using a standard AFM

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca2+-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (‘oligoGs’) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4 Ca2+ ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level

Sulfated Alginates as Heparin Analogues: A Review of Chemical and Functional Properties

Heparin is widely recognized for its potent anticoagulating effects, but has an additional wide range of biological properties due to its high negative charge and heterogeneous molecular structure. This heterogeneity has been one of the factors in motivating the exploration of functional analogues with a more predictable modification pattern and monosaccharide sequence, that can aid in elucidating structure-function relationships and further be structurally customized to fine-tune physical and biological properties toward novel therapeutic applications and biomaterials. Alginates have been of great interest in biomedicine due to their inherent biocompatibility, gentle gelling conditions, and structural versatility from chemo-enzymatic engineering, but display limited interactions with cells and biomolecules that are characteristic of heparin and the other glycosaminoglycans (GAGs) of the extracellular environment. Here, we review the chemistry and physical and biological properties of sulfated alginates as structural and functional heparin analogues, and discuss how they may be utilized in applications where the use of heparin and other sulfated GAGs is challenging and limited