Facilitating high-force single-polysaccharide stretching using covalent attachment of one end of the chain

Single polysaccharide force spectroscopy has yielded particularly interesting data, the interpretation of which requires the marriage of statistical-mechanical theories of polymer physics to the complexities afforded by possible force-induced conformational transitions of the constituent sugar rings. However, the difficulty of designing handles for the specific attachment of the different ends of polysaccharide chains to substrates, such as piezoelectric scanners, cantilevers or microbeads has meant that the majority of studies to date have been carried out with the polymer physisorbed to the substrates between which it is stretched, or at best chemically attached via bonds formed at uncontrolled locations along the length of the molecule. This means that the lengths of obtained polysaccharide stretches, as well as the forces that can be placed on the molecule without generating detachment, are generally smaller than those obtainable for polymers that offer the ability to be covalently attached to substrates specifically at their ends. As a consequence it is troublesome and tedious to record a statistically significant number of force curves that extend chains to high enough forces to investigate certain conformational transitions, such as the boat-to-inverted chair, exhibited by polysaccharides such as pectin. Herein, single molecule force-extension curves have been measured for the several pectin samples using AFM. The results are compared when either (1) the polymers have been physisorbed between the cantilever and the surface of the piezo-electric scanner, under several different solvent conditions of pH and ionic strength, or (2) the polymer molecule has been chemically attached at one end to the piezo surface using a recently reported coupling procedure. In fact, using such a chemical attachment to tether the end of the polysaccharide, reduced the frequency of successful stretching events obtained in a particular location, confirming the role of surface diffusion in the physisorbed experiments. Nevertheless, when polymer stretches were successfully recorded, the force that could be applied before detachment was significantly increased, indicating that this methodology has great potential for improving the acquisition of data reporting on force-induced conformational transitions of the sugar ring that require the application of significant stresses.