Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration

Abstract

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water–collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H 2 O/D 2 O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H 2 O and D 2 O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D 2 O than in H 2 O, and collagen in D 2 O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H 2 O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D 2 O is less hydrated than in H 2 O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen–water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.

Bioarchitectural Design of Bioactive Biopolymers: Structure–Function Paradigm for Diabetic Wound Healing

“…Its tertiary structure comprises three polypeptide chains (mainly composed of amino acids like proline, hydroxyproline, and glycine) wrapped in a left-handed triple helix to form collagen fibrils [190]. The process of collagen fibrillogenesis is strongly driven by increased entropy, resulting in a greater level of disorder at the water-protein interface [191]. Fibroblasts synthesize collagen de novo an integral part of normal wound healing process [192].…”

Is Deuterium Fractionation a Major Controlling Factor in Human Metabolism and Cancer? An Essential Role for Proline

“…Water forms bridges that contribute to the stability of the triple helix. It creates a repulsive interaction between collagen molecules, since the water bridges must be reorganized in order for the collagen molecules to get close to each other [55]. If the water is replaced by heavy water, the assembly of collagen occurs ten times faster.…”

“…If the water is replaced by heavy water, the assembly of collagen occurs ten times faster. This is due to the lowering of the energy penalty incurred by displacing the water bridges, which enhances the initial nucleation rate [55]. It is plausible that the enrichment in deuterium as a consequence of the battery formation accelerates collagen cross-link formation and assembly in the extracellular space.…”

Heterogeneous Structure and Dynamics of Water in a Hydrated Collagen Microfibril

“…19−21 Heavy water (D 2 O) has also been used as a way to fine-tune hydration of fibrils modulating hydrogen bonding, which resulted in lower hydration and faster self-assembly. 22 Temperature has also been used to trigger structural changes in collagen fibrils and influence intermolecular forces. 23 Beyond structural aspects, water dynamics within the fibril have received no or at most very little attention.…”

“…Experimental studies were therefore limited on water contents mostly ranging from dry to 80% water-to-protein mass ratio, which correspond to a full range of relative humidity (0% to 100%). In addition to intermolecular forces, swelling of the fibrils was characterized using AFM. − Heavy water (D 2 O) has also been used as a way to fine-tune hydration of fibrils modulating hydrogen bonding, which resulted in lower hydration and faster self-assembly . Temperature has also been used to trigger structural changes in collagen fibrils and influence intermolecular forces …”