Contributed Talk - Friday, 17 September I 10:50 AM (CEST)
Andreas Rohatschek: "AFM SMFS of individual collagen molecules"
TU Wien, Austria
Introduction Collagens are the most abundant and structurally the most important proteins of the human extracellular matrix. Therefore, mechanical properties of collagen molecules (tropocollagen), and the progressively larger structures they form, are crucial for tissue mechanics and function. While there are a number of studies modeling the mechanical behavior of tropocollagen molecules via molecular dynamics (MD) approaches there is little but none experimental data available. Due to MD limitations experimental validation of predicted behavior such as molecular uncoiling or interaction between neighboring molecules is needed and will also provide further insight tropocollagen mechanics. Here, we present an approach to experimentally characterize adhesion forces between tropocollagen molecules to substrates of choice. Methods Surface functionalization  is used to tether individual tropocollagen molecules onto an AFM tip. In a first step a polyethylene glycol (PEG) based linker system with N-hydroxysuccinimide (NHS) and maleimide (MI) coupling groups (NHS-PEG-MI) was coupled to an aminated Si AFM tip. Subsequently, collagen Type III was attached using the thiol group of cysteine by MI coupling. Atomic Force Microscopy (AFM) Single Molecule Force Spectroscopy (SMFS) measurements were conducted in different media. i.e. water, acetic acid, DPBS, CAPS and various substrates (glass, mica, TiO2) at different retraction speeds (100, 250, 500,1000, 1500 and 2000 nm/s) to investigate Tropocollagen-substrate interactions Results We observed a profound dependency of collagen substrate interactions on substrate type and surrounding medium. In a typical force curve, unspecific adhesion interactions are witnessed within the first 30 nm from the substrate surface due to tip-substrate interaction. Following this unspecific region tropocollagen, related interaction are observed up to pulling lengths of almost 800 nm (see Table 1). For measurements in acetic acid (0.5 mol/L), the determined adhesion energies for all retraction velocities lied between 1.05*e-17 J and 3.03*e-17 J (see Table 2). Discussion All measurements reproducibly show a similar interaction behaviour with the substrate used. Furthermore, pulling lengths corrected by the linker contribution (approx. 100 - 150 nm ) were detected within a plausible range. The determined median length suggests that uncoiling of entangled configuration (e.g. telopeptide region) and uncurling of the collagen Polyproline-II helix takes place . The successful measurements enable investigating collagen adhesion to a number of substrates including other collagens, and are an important step towards future tensile tests of individual tropocollagen molecules.
References 1. Kamruzzahan et. al. (2006), Bioconjugate Chemistry, 17(6), 1473–1481, https://doi.org/10.1021/bc060252a 2. Steinbauer, P et. al. (2020). Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein-Hydroxyapatite Interface. Langmuir, 36(44), 13292–13300. https://doi.org/10.1021/acs.langmuir.0c02325 3. Buehler, M. J., & Wong, S. Y. (2007). Entropic elasticity controls nanomechanics of single tropocollagen molecules. Biophysical Journal, 93(1), 37–43. https://doi.org/10.1529/biophysj.106.102616