Periodic Reporting for period 2 - RADICOL (Mechanoradicals in Collagen)
Reporting period: 2022-12-01 to 2024-05-31
Our tissues, in particular collagen as the most abundant protein in our body, are constantly exposed to mechanical loads, reaching multiples of the body weight. In artificial polymers, mechanical loads are known for a century to cause radical formation and chemical degradation processes. Mechanoradicals from bond ruptures, being highly reactive and oxidising, deteriorate the material, leading to stiffening and ageing. Ageing of organic tissue is a fundamental problem in health and disease, but a role of mechanoradicals has been a blind spot. Our simple but novel idea is to test the role of mechanoradicals for ageing of biomaterials. As a starting point, we have recently uncovered mechanoradicals in tensed tendon collagen. They readily react with water to form reactive oxygen species (ROS), key signalling molecules in a multitude of physiological processes including ageing.
Our aim is to test the hypothesis that mechanoradicals generate a feedback loop resulting in accelerated collagen ageing. Using a scale-bridging combined computational and experimental approach, we dissect the full lifecycle of mechanoradicals in collagen, from bond scission and radical migration to ROS formation, to uncover new mechanisms of radical-mediated ageing. Our methods include quantum chemical calculations and Molecular Dynamics (MD) simulations, and a new reactive Monte Carlo/MD scheme based on machine learned reaction barriers, in order to identify scissile bonds and subsequent radical reactions in atomistic collagen I fibril models. For validation, a combination of electron-paramagnetic resonance spectroscopy, mass spectrometry and other biophysical experiments is employed to measure degradation pathways, radicals and ROS under varying crosslink densities and types as present in young, aged and diseased tendon tissues.
RADICOL will establish protein mechanoradicals as an as yet uncovered source of oxidative stress, and as a new paradigm of biological mechanosensation and ageing.
Our aim is to test the hypothesis that mechanoradicals generate a feedback loop resulting in accelerated collagen ageing. Using a scale-bridging combined computational and experimental approach, we dissect the full lifecycle of mechanoradicals in collagen, from bond scission and radical migration to ROS formation, to uncover new mechanisms of radical-mediated ageing. Our methods include quantum chemical calculations and Molecular Dynamics (MD) simulations, and a new reactive Monte Carlo/MD scheme based on machine learned reaction barriers, in order to identify scissile bonds and subsequent radical reactions in atomistic collagen I fibril models. For validation, a combination of electron-paramagnetic resonance spectroscopy, mass spectrometry and other biophysical experiments is employed to measure degradation pathways, radicals and ROS under varying crosslink densities and types as present in young, aged and diseased tendon tissues.
RADICOL will establish protein mechanoradicals as an as yet uncovered source of oxidative stress, and as a new paradigm of biological mechanosensation and ageing.
During the past 30 months of the project, we reached a number of important milestones. Those include a detailed analysis of the radical scavenging capacity of DOPA in biochemical and chemical assays and computations and a quantum chemical assessment of bond dissociation energies in proteins as a basis of hydrogen atom migration calculations. An important result has been that we find DOPA indeed to be not only thermodynamically a stable radical but to outperform its precursors, tyrosine and phenylalanine, in this function. DOPA also very readily converts to the quinone resulting in the formation of hydrogen peroxide, all of which we could show by very specific biochemical assays supported by calculations. Another step forward has been the prediction of bond rupture sites by our multi-scale simulation approach, which we also could now validate by mass spectrometry. A key result is that collagen I of tendon ruptures primarily within or just next to crosslinks (see Figure). The trivalent crosslink shows one particularly weak link, the rupture of which leads to a situation in wihch the two triple helices are still connected. We thus termed this bond a sacrificial bond, which is a fully new concept for collagen.
All of our simulation work is based on our structural model of collagen, which we made available publicly and which includes a number of chemically different crosslinks as well as different collagen sequences. Taken together, we could establish collagen as a redox-active material. It tailors ruptures such that they are funneled into particularly weak links, where radicals can be readily scavanged through DOPA residues in the vicinity. Our results suggest collagen not to act as a mere mechanically important material, a biological rubber band, but to instead function as a 'mega-enzyme', in which mechanically triggered chemistry happens very specifically.
All of our simulation work is based on our structural model of collagen, which we made available publicly and which includes a number of chemically different crosslinks as well as different collagen sequences. Taken together, we could establish collagen as a redox-active material. It tailors ruptures such that they are funneled into particularly weak links, where radicals can be readily scavanged through DOPA residues in the vicinity. Our results suggest collagen not to act as a mere mechanically important material, a biological rubber band, but to instead function as a 'mega-enzyme', in which mechanically triggered chemistry happens very specifically.
Our studies so far laid the basis for the next steps towards addressing these questions as a function of collagen ageing, both in simulations using unspecific age-related or DOPA-related crosslinks, and in experiments using tissues from young and aged animals. We also have proceeded on developing KIMMDY, a reactive combined Monte Carlo / Molecular Dynamics simulation scheme, which we aim to finalize in the coming months in a first version to be released publicly. In this version, users will be able to simulate covalent bond rupture and radical migration. Radical recombination and other types of bond rupture, by heterolysis, remain to be included. KIMMDY will be published with examples beyond collagen, such as small peptides as well as large synthetic polymer systems.
OVerall, the project will lead to a first quantitative view on how mechanoradicals are formed and processed by collagen, and how collagen has been designed to cope with them, shedding new light on tissue ageing and disease.
OVerall, the project will lead to a first quantitative view on how mechanoradicals are formed and processed by collagen, and how collagen has been designed to cope with them, shedding new light on tissue ageing and disease.