Periodic Reporting for period 1 - CatchGel (Catch Bond Cross-linked Hydrogels)
Periodo di rendicontazione: 2020-10-01 al 2023-09-30
Catch bonds, in contrast to traditional slip bonds, are protein complexes that exhibit an increase in the rupture force of the complex under applied mechanical load. Although highly dependent on the type of catch bond, the increase in rupture forces can be up to 10-fold. However, this has only been observed on the molecular scale. What if we could exploit this behaviour in a macroscale material? This would be a major advantage for materials science, particularly for materials exposed to dynamic mechanical stress, such as in trauma care or regenerative medicine. In particular in regenerative medicine there is a need for materials that adapt their viscoelastic properties dynamically to mechanical/biochemical cues from the cellular environment. Materials cross-linked by catch bonds complexes, that were weaker at equilibrium but became stiffer as applied forces increased, could be advantageous for the creation of dynamic substrates on which to culture tissues and organs, or organoids, or to address rapidly evolving medical situations, for example in trauma care. But effectively translating these nanoscale behaviours to the macroscale requires careful study of the limits of stability of these complexes, the role of the environment, and the types of stress both at the molecular scale and as we expand towards the macroscale. The goal of this project was to use detailed biophysical and mechanical characterisation methods across multiple length scales to develop an approach to transplant catch bonding protein complexes into macroscale materials, while preserving their unique mechanosensitive properties.
The work plan was divided into three main areas; (1) identify and characterise a selection of catch bonding protein complexes of interest, (2) develop a mild, site-specific methodology to functionalise polymer scaffolds with complexes of interest, and (3) to characterise the resulting hydrogels and their adaptability to mechanical stress and environmental changes.
Serine-aspartate repeat protein G (sdrG) from S. epidermidis and its complex with the beta strand of the extracellular matrix (ECM) protein fibrinogen (FgB), as well as the complex between pilus-related adhesin A (RrgA) from S. pneumoniae and the ECM protein fibronectin (Fn) were initially chosen as catch bonding (CB) complexes of interest, representing one of the strongest (sdrG-FgB) and one of the weakest (RrgA-Fn) bacterial CB complexes known at the time. However, initial experiments identified issues with RrgA-Fn as a model complex due to the weakness of the rupture forces and the similarity of the low and high force rupture regimes, which were more dramatically different in the sdrG-FgB complex. This was deemed to be more useful for eventual tuning of macroscale materials. Thus, focus was turned to exploring the limits of adaptability of sdrG-FgB and the effect of geometric and biochemical modifications to the sdrG structure, environmental and mechanical stress using single molecule force spectroscopy (SMFS). Here, we identified a strong effect of calcium concentration, geometric arrangement and point mutations, as well as the type of substrate (if we isolated the beta strand or used the full Fg protein as the complement) on the behaviour of the complex, while the expected dependence on pulling speed was remarkably small. This encouraged us to look more closely at cooperativity between the protein complexes because cells expressing CB proteins do have the ability to stick stronger as a function of applied force, but why not the individual proteins to such a great extent?
Instead of moving directly to the macroscale, we displayed the sdrG and its various mutants on the surface of yeast cells and used a technique called spinning disk microscopy (SDM), to apply shear stress to the cells after incubation on an Fg or FgB substrate and look at the cell distribution on the glass substrate as a function of shear force to determine the adhesive behaviour of the individual complexes. Here we saw the shear dependent behaviour we were hoping for, but this indicated that perhaps the sticking ability of pathogenic bacteria is a cooperative rather than a single molecule effect, something we hadn’t previously expected! We followed this up with particle imaging velocimetry and numerical modelling using computational fluid dynamics to further develop this theory, which further confirmed the importance of cooperativity but downplayed the importance of geometry at the microscale.
Finally to the macroscale. We successfully developed a mild methodology to ensure site-specific immobilisation of the proteins on aminated polysaccharides (primarily chitosan, but we were also able to aminate other polysaccharides and achieve functionalisation of the surface with the proteins) with reasonable reproducibility (issues with reproducibility were more linked to the variations observed in polymers of natural origin). We also explored embedding yeast cells with displayed proteins or protein-functionalised microspheres in inert polysaccharide matrices of alginate or gellan gum. This gave us detailed insight into the importance of the polysaccharide backbone; a weaker or more flexible polysaccharide made it easier to see the effect of the CB cross-links but gave us a material that was difficult to manipulate, while a more robust backbone gave better handleability but dampened the effect of the catch bond. A lot of time was spent optimising the perfect CatchGel until it was clear there was no perfect option, just the optimum for a specific application. Mass photometry, rheology, and AFM-microrheology were all used to come to this conclusion.
In summary, by employing single molecule force spectroscopy, spinning disk microscopy, rheology and a number of ancillary techniques to verify our observations, alongside numerical modelling we were able to investigate complex multi-lengthscale interactions and elucidate design parameters for the exploitation of mechanosensitive protein complexes on the macroscale.
Serine-aspartate repeat protein G (sdrG) from S. epidermidis and its complex with the beta strand of the extracellular matrix (ECM) protein fibrinogen (FgB), as well as the complex between pilus-related adhesin A (RrgA) from S. pneumoniae and the ECM protein fibronectin (Fn) were initially chosen as catch bonding (CB) complexes of interest, representing one of the strongest (sdrG-FgB) and one of the weakest (RrgA-Fn) bacterial CB complexes known at the time. However, initial experiments identified issues with RrgA-Fn as a model complex due to the weakness of the rupture forces and the similarity of the low and high force rupture regimes, which were more dramatically different in the sdrG-FgB complex. This was deemed to be more useful for eventual tuning of macroscale materials. Thus, focus was turned to exploring the limits of adaptability of sdrG-FgB and the effect of geometric and biochemical modifications to the sdrG structure, environmental and mechanical stress using single molecule force spectroscopy (SMFS). Here, we identified a strong effect of calcium concentration, geometric arrangement and point mutations, as well as the type of substrate (if we isolated the beta strand or used the full Fg protein as the complement) on the behaviour of the complex, while the expected dependence on pulling speed was remarkably small. This encouraged us to look more closely at cooperativity between the protein complexes because cells expressing CB proteins do have the ability to stick stronger as a function of applied force, but why not the individual proteins to such a great extent?
Instead of moving directly to the macroscale, we displayed the sdrG and its various mutants on the surface of yeast cells and used a technique called spinning disk microscopy (SDM), to apply shear stress to the cells after incubation on an Fg or FgB substrate and look at the cell distribution on the glass substrate as a function of shear force to determine the adhesive behaviour of the individual complexes. Here we saw the shear dependent behaviour we were hoping for, but this indicated that perhaps the sticking ability of pathogenic bacteria is a cooperative rather than a single molecule effect, something we hadn’t previously expected! We followed this up with particle imaging velocimetry and numerical modelling using computational fluid dynamics to further develop this theory, which further confirmed the importance of cooperativity but downplayed the importance of geometry at the microscale.
Finally to the macroscale. We successfully developed a mild methodology to ensure site-specific immobilisation of the proteins on aminated polysaccharides (primarily chitosan, but we were also able to aminate other polysaccharides and achieve functionalisation of the surface with the proteins) with reasonable reproducibility (issues with reproducibility were more linked to the variations observed in polymers of natural origin). We also explored embedding yeast cells with displayed proteins or protein-functionalised microspheres in inert polysaccharide matrices of alginate or gellan gum. This gave us detailed insight into the importance of the polysaccharide backbone; a weaker or more flexible polysaccharide made it easier to see the effect of the CB cross-links but gave us a material that was difficult to manipulate, while a more robust backbone gave better handleability but dampened the effect of the catch bond. A lot of time was spent optimising the perfect CatchGel until it was clear there was no perfect option, just the optimum for a specific application. Mass photometry, rheology, and AFM-microrheology were all used to come to this conclusion.
In summary, by employing single molecule force spectroscopy, spinning disk microscopy, rheology and a number of ancillary techniques to verify our observations, alongside numerical modelling we were able to investigate complex multi-lengthscale interactions and elucidate design parameters for the exploitation of mechanosensitive protein complexes on the macroscale.
The conclusion of this project brought with it several areas in which progress beyond the state-of-the-art was achieved. The highlights of this project have been a detailed insight into the molecular behaviour of our selected adhesin complexes, a molecular to microscale understanding of single molecule vs cooperative protein complex behaviours, a successful methodology for transplanting catch bond forming complexes into macroscale materials and application of these materials beyond the scope of the project. Knowledge obtained in this project facilitated funding acquisition for two spin-off projects in the direction of impact resistant materials and dynamic cell culture matrices. This project has advanced our fundamental knowledge of catch bonds, their scalability and their sensitivity to environmental stress, and allowed us to develop materials with great potential for socio-economic impact in the area of regenerative medicine and medical technologies.