article published yes Giulia Giubertoni author Liru Feng author Kevin Klein author Guido Giannetti author Luco Rutten author Yeji Choi author Anouk Van Der Net author Gerard Castro-Linares author Federico Caporaletti author Dimitra Micha author Johannes Hunger author Antoine Deblais author Daniel Bonn author Nico Sommerdijk author Anđela Šarić author bf63d406-f056-11eb-b41d-f263a6566d8b0000-0002-7854-2139 Ioana M. Ilie author Gijsje H. Koenderink author Sander Woutersen author AnSa department 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 H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H2O and D2O 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 D2O than in H2O, and collagen in D2O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. 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. https://research-explorer.ista.ac.at/download/15116/15125/2024_PNAS_Giubertoni.pdf application/pdfno https://creativecommons.org/licenses/by/4.0/ National Academy of Sciences2024 eng 0027-8424 1091-6490 38451946 00120638740000110.1073/pnas.2313162121 12111 https://research-explorer.ista.ac.at/record/15126 Giubertoni, Giulia, et al. “Elucidating the Role of Water in Collagen Self-Assembly by Isotopically Modulating Collagen Hydration.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 121, no. 11, e2313162121, National Academy of Sciences, 2024, doi:<a href="https://doi.org/10.1073/pnas.2313162121">10.1073/pnas.2313162121</a>. Giubertoni G, Feng L, Klein K, et al. Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. 2024;121(11). doi:<a href="https://doi.org/10.1073/pnas.2313162121">10.1073/pnas.2313162121</a> Giubertoni, Giulia, Liru Feng, Kevin Klein, Guido Giannetti, Luco Rutten, Yeji Choi, Anouk Van Der Net, et al. “Elucidating the Role of Water in Collagen Self-Assembly by Isotopically Modulating Collagen Hydration.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences, 2024. <a href="https://doi.org/10.1073/pnas.2313162121">https://doi.org/10.1073/pnas.2313162121</a>. G. Giubertoni <i>et al.</i>, “Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration,” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 121, no. 11. National Academy of Sciences, 2024. Giubertoni G, Feng L, Klein K, Giannetti G, Rutten L, Choi Y, Van Der Net A, Castro-Linares G, Caporaletti F, Micha D, Hunger J, Deblais A, Bonn D, Sommerdijk N, Šarić A, Ilie IM, Koenderink GH, Woutersen S. 2024. Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration. Proceedings of the National Academy of Sciences of the United States of America. 121(11), e2313162121. Giubertoni, G., Feng, L., Klein, K., Giannetti, G., Rutten, L., Choi, Y., … Woutersen, S. (2024). Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences. <a href="https://doi.org/10.1073/pnas.2313162121">https://doi.org/10.1073/pnas.2313162121</a> G. Giubertoni, L. Feng, K. Klein, G. Giannetti, L. Rutten, Y. Choi, A. Van Der Net, G. Castro-Linares, F. Caporaletti, D. Micha, J. Hunger, A. Deblais, D. Bonn, N. Sommerdijk, A. Šarić, I.M. Ilie, G.H. Koenderink, S. Woutersen, Proceedings of the National Academy of Sciences of the United States of America 121 (2024). 151162024-03-17T23:00:57Z2025-09-04T13:03:56Z