Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM

Abstract

Under a stretching force, the sugar ring of polysaccharide molecules switches from the chair to the boat-like or inverted chair conformation. This conformational change can be observed by stretching single polysaccharide molecules with an atomic force microscope. In those early experiments, the molecules were stretched at a constant rate while the resulting force changed over wide ranges. However, because the rings undergo force-dependent transitions, an experimental arrangement where the force is the free variable introduces an undesirable level of complexity in the results. Here we demonstrate the use of force-ramp atomic force microscopy to capture the conformational changes in single polysaccharide molecules. Force-ramp atomic force microscopy readily captures the ring transitions under conditions where the entropic elasticity of the molecule is separated from its conformational transitions, enabling a quantitative analysis of the data with a simple two-state model. This analysis directly provides the physico-chemical characteristics of the ring transitions such as the width of the energy barrier, the relative energy of the conformers, and their enthalpic elasticity. Our experiments enhance the ability of single-molecule force spectroscopy to make high-resolution measurements of the conformations of single polysaccharide molecules under a stretching force, making an important addition to polysaccharide spectroscopy. S ingle molecule force spectroscopy has become an important tool to examine the conformations of proteins and polysaccharide molecules under a stretching force (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Recent experiments have demonstrated that a force field can trigger conformational changes in these molecules that cannot be observed by traditional NMR or x-ray crystallographic techniques. Force spectroscopy involves measuring the force required to extend a molecule by a certain amount (2-10). The data are compiled into a forceextension relationship that gives a characteristic fingerprint for the conformational transitions of the molecule under study. For example, force-extension relationships of polysaccharides such as amylose and dextran (13, 14) display a characteristic plateau (2, 4) that marks forced transitions of the glucose monomers from their chair conformation to the boat-like conformation (4). Similarly, two distinct plateaus in the force-extension curve of pectin (13, 14) report a transition of the galactose rings from the chair conformation to the boat conformation and a subsequent flip of the boat to the inverted chair conformation (5). These observations provide an interesting perspective on the behavior of polysaccharides under mechanical tension (15) and suggest that fingerprints of forced conformational transitions can be used as a means for identifying single polysaccharides molecules in solution (9).In the most typical atomic force microscopy (AFM) experiment, a single molecule adsorbed between a substrate and the cantilever tip is extended vertically at a constant rate while t...

Puckering free energy of pyranoses: A NMR and metadynamics-umbrella sampling investigation

“…The estimate obtained from CMA, however, could be biased because of the inhability of a freely jointed chain model to fit the force-extension curves of CMA, which prefers a pseudo-helical conformation, as it has been pointed out already in Ref. 66. Recently, Kuttel and Naidoo 70 suggested that in amylose stretching the elongation might be due to a complex rotation of glycosidic linkages, and that chair to boat transitions could play little role in the process.…”

“…Marszalek, Lee, and coworkers 45,66,67 , for example, estimated the puckering free energy of glucose twisted boats employing AFM pulling on dextran, cellulose, and pustulan.…”

Can Dissipative Properties of Single Molecules Be Extracted from a Force Spectroscopy Experiment?

“…Of course, these two pairs of signals (viz. X,Y and amplitude and phase signals) are not independent but are related to each other according to the formulae (15)(16)(17)(18). A typical lock-in amplifier does not enable one to record both signal pairs simultaneously, but of course they can be restored (calculated) from each other, as illustrated in Fig.…”

“…Experiments with dextran were performed in ultrapure water. It is known that dextran molecules are not charged, and water represents a good solvent for force spectroscopy experiments in this case (18,19), similarly to media such as phosphate-buffered saline and ethanol. For the latter, it has been shown that the elastic response of dextran is not influenced by changing buffer conditions (19).…”

“…Dextran and I27 protein molecules were chosen for these starting experiments because both of these molecules can be regarded as standard (if not calibrating) samples for single-molecule force spectroscopy, and have been widely studied (see, e.g., (8,13,18,19,(23)(24)(25)(26)(27)(28)(29)(30)). The dissipative properties of both of these molecules have been described elsewhere (8,13).…”