Differences in zero-force and force-driven kinetics of ligand dissociationfrom beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule forcemicroscopy

Protein-carbohydrate interactions are involved in diverse regulatory proces ses, To help understand the mechanics and kinetics of dissociation of recep tor-ligand complexes, we have analyzed the separation of lactose and the N- glycan chains of asialofetuin (ASF) from three lectins and an immunoglobuli n G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)(2) agglut inins from Ricinus communis (RCA) and Viscum album (VAA) show structural ho mology, the homodimeric galectin-1 from bovine heart (BHL) has no similarit y to the two plant lectins except for sharing this monosaccharide specifici ty. The beta -galactoside-binding immunoglobulin G (IgG) fraction from huma n serum provides a further model system with distinct binding-site architec ture, The k(off) constants for the two plant agglutinins were independent o f the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0. 5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG: (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing c omparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/ - 9 pN (RCA) were measured, For the same loading rate the rupture forces fo r the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupt ure force and the natural logarithm of the loading rate, performing probabi lity density and Monte Carlo calculations, the potential barrier widths, wh ich determine the inverse dynamic dependence with the rate of force elevati on, increased from 4 Angstrom (RCA) and 7 Angstrom (VAA and IgG) to 10 Angs trom (BHL) for the receptor-lactose interactions. Presenting ASF as ligand potential widths of 4 Angstrom for RCA and IgG and 6 Angstrom for VAA and B HL were obtained. Since the dissociation kinetics at zero force apparently cannot predict the behavior in force-driven experiments, these results reve al new insights into biological functions, The dissociation kinetics under force helps to explain the difference in the toxic potency of VAA and RCA a nd points to a function of the galectin in cis-crosslinking and in transien t trans-bridging. (C) 2000 Academic Press.