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

Maintaining dimensions and mechanical properties of ionically crosslinked alginate hydrogel scaffolds in vitro

Ionically crosslinked alginate hydrogels are attractive scaffolds because of their biocompatibility and mild gelation reaction that allows for gentle cell incorporation. However, the instability of ionically crosslinked hydrogels in an aqueous environment is a challenge that limits their application. This report presents a novel method to control the dimensions and mechanical properties of ionically crosslinked hydrogels via control of the ionic concentration of the medium. Homogeneous calcium-alginate gels were incubated in physiological saline baths adjusted to specific calcium ion concentrations. Swelling and shrinking occurred at low and high ionic concentrations of the medium, respectively, while an “optimal” intermediate calcium ion concentration of the medium was found to maintain original size and shape of the hydrogel. This optimal calcium ion concentration was found to be a function of crosslinking density and polymer concentration of the hydrogel and chemical composition of the alginate. The effects of optimal and high calcium ion concentrations of the medium on swelling behavior, calcium content, dry weight, and mechanical properties of the immersed hydrogels were investigated. It was found that the resulting hydrogel composition and mechanical properties depended on not only the calcium concentration of the medium, but also the crosslinking density and polymer concentration of the gel. In an 8-week experiment, controlled dimensions and mechanical properties of alginate gels in an aqueous environment were demonstrated. This new technique significantly enhances the potential of alginate hydrogels for tissue engineering and other biomedical applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58038/1/31375_ftp.pd

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

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

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

Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.Cystic Fibrosis Foundation (Research Development Program)National Center for Research Resources (U.S.) (P20RR018787-06

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors

Lentivectors are widely used for gene delivery and have been increasingly tested in clinical trials. However, achieving safe, localized, and sufficient gene expression remain key challenges for effective lentivectoral therapy. Localized and efficient gene expression can be promoted by developing material systems to deliver lentivectors. Here, we address the utility of microgel encapsulation as a strategy for the controlled release of lentivectors. Three distinct routes for ionotropic gelation of alginate were incorporated into microfluidic templating to create lentivector-loaded microgels. Comparisons of the three microgels revealed marked differences in mechanical properties, crosslinking environment, and ultimately lentivector release and functional gene expression in vitro. Gelation with chelated calcium demonstrated low utility for gene delivery due to a loss of lentivector function with acidic gelation conditions. Both calcium carbonate gelation, and calcium chloride gelation, preserved lentivector function with a more sustained transduction and gene expression over 4 days observed with calcium chloride gelated microgels. The validation of these two strategies for lentivector microencapsulation may provide a platform for controlled gene delivery