An Atomistic View On Protein Adsorption to Interfaces and the Importance of Wettability on Drop Break-up in a Capillary Confinement from a Fluid-Dynamic Perspective
Giefer, Patrick
Emulsions are widely used in a variety of different industries and applications with increasing importance. Two main objectives need to be addressed in the emulsion formulation: The emulsion needs to be formed with a narrow and predefined size distribution and it needs to be stabilized to prevent coalescence and therefore facilitate handling of the emulsion. So far the breakup mechanism of droplets in Membranes is still unknown. Further the role of proteins on breakup mechanism due to their interfacial adsorption and induces change in wettability needs to be addressed. In this work Molecular Dynamic simulations were performed to give an insight on protein adsorption to oil/water and water/SIO2 interfaces to understand which role Proteins play in this context. Furthermore Computational Fluid Dynamics were conducted to clarify the breakup mechanism in capillary confinements under varying fluid properties as well as membrane wettability. The results show that the proteins not only adsorb to fluid/fluid but also fluid/solid interfaces and therefore change the properties of the membrane. From the fluid-dynamic perspective membrane wettability plays a major role on the droplet dispersion as well as in the emergence of fluid dynamic instabilities that eventually lead to breakup.
 Thank you very much for the introduction. I apologize the long title. I didn't know how to combine these two areas. I guess most of you were asked themselves, like, why proteins now and probably don't have any connection, academic connection to proteins, but I'm sure you maybe already consume them this morning at your breakfast. With this I would like to introduce into the industrial relevance at the example of milk home organization. So milk is a multi-phase system which basically contains water, oil, and proteins. And what happens in milk home organization, you force, you apply some stress, you force it through membrane, for example, and then you create fine monodysperous emulsion. And on the right hand side I would like to show you that there's a schematic plot of the stress residence time. So you can have a long residence time of those proteins and those droplets in the system with a relatively low shear stress. You can have medium exposure to shear stress and time. And you can have long and short and high stress peaks. So the residence time, the stress rem, and the level of stress are very important factors for this kind of process. And the question is then what is the influence on drop breakup itself and break up and protein degradation. The motivation comes from the premix membrane emulsion. So you basically start with a rough premix. It's already water, oil, and proteins. You force it with a pressure gradient through a membrane to create some fine and monodysperous emulsions. And how this works is due to shear and strain stress inside the membrane. And the hypothesis is that the protein absorption affects the wetability of those membranes and which also affect then the break up mechanism. I would like to look at two different scales today, the macro and micro scale, which is the fluid dynamics, so the droplets version inside the membrane, the influence of the wetability on the droplets version mechanism inside. And on the other scale, the atomic scale with molecular dynamic simulations, the adsorption of the protein as at the fluid fluid interface. So it basically adopts to the fluid interface to reduce interfacial tension there and therefore stabilize emulsions. And also the adsorption to fluid solid interfaces as in experiments we have seen membrane clogging, so protein adoption to the membrane itself. To start, I would like to talk about the influence on the drop dispersion mechanism. And we identified the wall region as a region of high shear stress, so we would like to have a closer look at the wall interactions. This is I accidentally got to the end. Right, so the drop dispersion in forest media is a very complex phenomenon. We have multiple semi-chains break up events. We have a complex and very irregular structure. And this leads to the fact that the break up mechanism inside of those membranes is pretty much unknown. What can we do to address this? We can reduce complexity, of course. We will look at the capillary break up, so we just have a single droplet in a single pour and we can quantify therefore influencing factors on the single break up mechanism. With the numerical setup, we use oil and water, so with the according fluid properties, our simulation parameters, we vary the contact angle in our simulations. The dimensions, we have a 200 micrometer pour, which is 6 millimeters long and we push a droplet of 500 micrometers into this pour with very capillary numbers as low as 1e-3 to 1 with the according Weber-Andrejnod's numbers. The mesh, we use a hexahedral mesh size over a mesh size of 12 micrometers and we refined it down to 3 micrometers at the interface with an adaptive mesh refinement. We keep a current Fridtisch-Lewis criteria as low as 0.5 to satisfy the advection of the fluid phase within our domain and we also keep this time step as low as necessary to resolve capillary waves to account for various velocities. Further, we use open foam and we use a solver called Interflow, so it's basically a volume of fluid approach. You have a phase fraction based interface capturing. This means you have a phase fraction that denotes the fluids. For example, one here would be the fluid A, zero would be the fluid B and in between 0.5 is where our interface is located. We solved the Navistock's equation with continuity and momentum equation. We applied for surface tension a continuum surface force model from Rekkel and Korten, but recently we also implemented a sharp surface tension force as these systems are so small that they are really affected by spurious velocities which occur from discretization errors and also errors from the implementation of the surface tension force. To account for the wall interaction, we use a dynamic contact angle model which includes the function of Kistler which basically tells us that if we increase the velocity, our contact angle will also increase. We use a full three-dimensional hexahedral mesh with interface refinement around 0.5. Here I would like to show you some examples of four different simulations from hydrophilic to hydrophobic membranes. We can first divide this into five different regions. We have a leading front of the polymers droplet. We have a leading contact line and we have a wetting region and a trailing contact line with the overall trailing side. We now only concentrate on the leading front. We can see in the top right figure that the wave length of the leading front instability is dependent on the wetability. We conducted linear stability analysis for this from Lenz and Kumar to explain this. This is basically a model which takes confined liquid film trial layers and calculates the growth rate and the dominant wave number according to the layer height because we could observe that for our hydrophobic membranes, our wall layer of continuous fluid was actually thicker than for the hydrophilic ones. The results only show that the growth rate is increasing but not the wave number is increasing. The question still remains how does the wetability affect the unstable modes of the leading front instability? One explanation is we saw that the first moment the droplet enters the pore and it touches the pore is really crucial part. You might expect that if a droplet enters the membrane, it would directly attach to the membrane itself. So we have one located but actually with higher capillary numbers, it enters the pore, it necks at the entrance and the interface advex to the wall at later deeper inside the pore. You could see that this necking is also dependent on the contact angle. So if the more hydrophobic the membrane is, so the better the oil wets the membrane, the bigger the wetting. And we could see that at point two, the interfacial advection to the wall has shown some radial velocity which was also increasing with increasing wall wetting of the oil droplet. The droplet now further emerges into the pore. The continuous phase eventually gets trapped between contact lines. And what happens then is we can explain it by some effect called capillary venturi. So it's basically a venturi effect inside a capillary where capillary forces and hydrodynamic forces stay in competition to each other. Since they stay in competition to each other, they will be drawn inside, this volume will be drawn inside of the capillary to the pore center, which on the right hand side you can see that due to this entrapped phase and due to a contact line instability, this fingering instability is formed on the right lower figure. In easier words, if we see the enclosed entrapped continuous phase as a droplet, we have a contact line velocity directly at the pore and we have a mean pore velocity inside the pore. And the gradient leads to the fact that the water droplet travels inside the pore and forms this water droplets in oil. Here I would just like to give you some regimes of those fingering instabilities. On the left hand side, we can see probably the most famous fingering instability, which is the displacement of more viscous fluid by a less viscous fluid. In the middle, we can see a fingering instability that's existing because of the receding unstable interface, so contact line instability. And on the right hand side, we can see a droplet dropped on the surface with a high impact velocity and from this rim surrounding the droplet, we can see fingers emerging. This to the fluid dynamics, now I would like to continue to the atomic scale with the molecular dynamics just to keep in mind, we look at the absorption of protein at the fluid fluid interface for the reduction of interfacial tension and therefore stabilizing the droplets. On the other hand, we look at absorption to fluid solid interfaces, so the membrane clogging we observed in experiments. I would like to look at the PASA immobilization and relate the experimental literature to it. Absorb PASA to Zilliqa has shown an increased activity and also has shown a specificity and an anchor selectivity. They count some lid residues of this protein for this modulation of the activity and the specificity. What do I mean with lid residues? So the PASA is not only protein, it's also an enzyme, so it has some functional groups and it has a lid which opens the functional groups and closes the functional groups. Our numerical domain looks something like this, so we have a bulk of water, we have our protein, we have an interface which is silica or oil, no case, and we have water again, because we are dealing with periodic boundary conditions. We have to equilibrate our system and some preliminary research we have to do as rotational analysis. The PASA is quite a chunky protein and we cannot simply cannot wait for it to be attracted by the surface itself, so we have to do some preliminary research in order to account for this issue. What we do is we put the protein, place it in proximity to the interface, then we run a static MD run where we just calculate one molecular dynamic step, we save the interaction forces between the protein and the interface, then we rotate it, keeping the distance constant to the interface, run the MD run, save the interaction forces, and what we then have is basically a sampling of the surface and the interaction potential of the protein to the interface and we end up with the most attractive position of the protein relative to the interface from which we can run dynamic simulations then. Just a quick estimate for 500 nanoseconds, we need like 84 hours on a 1000 CPU cores, so we cannot really reach time scales, we can reach in fluid dynamics and this I will also address shortly. So I already talked about activation and increased activation due to adsorption, here I want to further clarify this fact on the left hand side, we can see the passive or inactive depasal conformation, I highlighted the lid residues in purple and if you compare from left to right you can see that these lid residues are in a different position, they expose on the right hand side the active center which is basically the place where the catalytic reaction happens inside the enzyme and they expose this part in the active conformation which then allows ligands actually to dock inside of this enzyme and for the enzyme to catalyze the reaction. We put our protein on surfaces, two different surfaces, in oil and on silica and what you can see here from the snapshot is basically that we have different adsorption configurations at liquid-liquid and liquid-solid interfaces, at liquid-liquid interfaces it adsorbs with the lid residues facing into the oil phase whereas on the right hand side the silica it exposes the lid residues to the water phase, I remove the water here because otherwise you wouldn't see anything, so just in case you're wondering. Excessibility of the active center and the lid residues for ligands on the silica adsorption. We look now further into the silica adsorption, we want to know what exactly happens, we could just see that it adsorbs so we can see by plotting the lid residues, each individual lid residues I plotted the distance between and on this graph we can see that at around 250 nanoseconds it starts to move and it starts to deviate from its equilibrium position which can be also confirmed by just looking at snapshots and highlighting the lid residues we can see it moves further to the active center. The question is what triggers the movement, we remember that the lid residues were physically distanced from the interface so there has to be something that triggers this movement close to the interface itself. For this we did some binding analysis and we basically took every residue of this protein and measured the distance between the interface and the residue and whenever it was in a distance where it could possibly build hydrogen bonds we marked this as binding and what you end up with is this kind of graph so you can see from this that you have constant binders which are basically constantly attached to the surface and frequent binders then would look more like this barcode structure. Interestingly we can see that the residue 522 lifts at around 250 nanoseconds from the surface which was previously a constant binder and which the result is the lid movement. We did some reference simulations for this so we just simulated the protein just in bulk water and we couldn't observe the lid's movement in these simulations. Further we did this also for the passive starting structure and there plotting the distance of the individual lid residues we couldn't see any lid movement whatsoever. So the question still remains is the protein now active or inactive? When we speak of interfacial activation or not and how can we address and determine protein activity numerically? First accessibility of the active center has to be guaranteed for a ligand actually to dock inside of this protein and second of all the ligand has to bind inside so the catalytic triad has to be still in a functional arrangement where it could possibly cut our ligand in pieces. To address the first issue we did some tunnel calculations with a tool called CAVA so what you basically do is you go from the surface of the protein and find tunnels which are connected from the outer surface to the catalytic triad and we can see on the left on the starting configuration that we have an open access to the active center and in the let's say natural case the ligands would just directly dock onto the protein and not even entering fully. On the right hand side we can see a complex tunnel network which has developed which is introduced by the bulk we confirm this in bulk simulations so the water introduces this tunnel but we can say that the tunnels are stabilized due to the energy minimization due to adsorption to this interface. Still active or inactive? Here I would like to show the catalytic triad what's the only thing you have to know is that it works together so it has to be in a functional arrangement of an acid base in a nucleophile and what happens is that a nucleophile in our case 0209 attacks our ligand with a nucleophilic attack to cut it. In our case we simulated a medium change triglyceride to address the questions if the catalytic triad is still in functional arrangement and how does it actually dock inside? This would in some give us an indication of the activity of the protein. We did this for a C8 triglyceride on the left hand side you can see that it docks into the starting configuration of the active protein and on the middle part you can see here that 0209 which is our nucleophile and our catalytic triad stabilizes this part of the ligand with hydrophobic interactions whereas for the adsorb structure we couldn't observe these interactions anymore so which would then give us an indication of the selectivity already of this protein. You varied the input structure, the docking structure and we did this with a C8, C6 and C10 chain and for the starting configuration we couldn't we could see that it was docking inside the protein but it was actually not interacting with the catalytic triad so it was just stabilizing this arrangement but not actually in a position where it could possibly catalyze the reaction. Whereas on the right hand side for the adsorb structure we could see that the C10 chain here is stabilized by our nucleophile. So in sum we have an indication of the selectivity and an ancilloselectivity and ancilloselectivity I forgot so we also put the enantiomere of this ligand inside the protein and docked it there and we also couldn't observe interactions with the adsorb structure and our ligand. Still this is a work in progress these are just some few examples which we have docked inside we are running a lot more in order to really tell what is this adsorb structure selective to and how can we determine for example which chain the experimentalists have to make longer or shorter in order for the adsorb structure to accept it or not. This I would like to come to the conclusion and to the outlook so the key findings from the computational fluid dynamics were that wetability affects the most unstable mode of the leading front instability that we have fingering in stability as a result of entrap continuous phase from molecular dynamics we could see that we have first of all different adsorption configurations of the parser liquid liquid and liquid solid interfaces. We have seen that adsorption of the parser to SU2 interfaces leads to an accessibility of the active center through a tunnel network which we could also imagine that we have an improved removal of the catalytic product through the tunnel network and our ligand binding simulations indicate different confirmations of the catalytic triad or the binding pockets. The key questions are how can we combine CFD and MD results as I said I agree pretty limited in the time space in the molecular dynamics and how can we combine these two things together we can quantify shear stress for example in computation fluid dynamics and then apply this shear stress to the molecular dynamics now that we found some stable configurations where our proteins adsorb where we can determine if it's active or inactive we can actually try now to destroy it in order to see to draw the whole picture what is the effect of forcing it with a pressure gradient through a membrane maybe we destroy the proteins the adsorpt structures maybe we don't and the last question is how does the adsorpt protein to liquid liquid liquid solid and liquid liquid solid as we have seen it could be possible that it adsorbs to to a solid liquid interface but then has still the the part exposed where it could adsorb to a fluid fluid interface so how does this influence properties like what ability in our use case this I would like to acknowledge a few people we also work for the molecular dynamics part with the hybrid materials and interfaces group of professor Colin B. Chuckley and yeah HLRN I would thank for computational resources and all of you for your attention and open for questions. Did you employ a special approaches for your protein folding also standard and the second one is have how about the force feed validation for interacting of the protein with the surfaces can you say how this was done in your force field model. So you mean validation from the interaction process is it standard. This is experimentally validated by the hybrid interface group of agreement so there is if you're interested in publication about the experimental work and the interaction forces between proteins and interfaces. In a similar direction but kind of potency use for the MD because you have different materials proteins and unorganic materials on your simulation. But for the unorganic materials as I said is done by the group by the hybrid materials and interfaces group they did some experiments and parameterized the force fields for it. And for the oil we can just use also slightly changed standard direction potentials. So for the MD part did you only look at the structural properties or did you consider something like a chart densities as well. Yes we did but I'm just starting to look into this first we were just focusing on the structures. Okay and for the interaction parts. Did you consider something like QMM approaches as well. Okay thanks. The contact and your model. Given the parameter or is it an unknown problem. So it's given parameter by the material we use but it's a dynamic in terms of velocity dependence so as the velocity increases and this is basically what the Kisla model says is that at the capillary number of one our contact angle would always increase towards 180 degrees. So what it does is it basically takes the velocity at the contact line and then calculates the capillary number there in order to change the direction of the surface force applied there to apply the contact angle. What is your boundary condition in the next two to the contact point? No slip boundary condition. Right now yes for this it was no slip but we currently implementing another slip condition in order to account for the non-integrable stress similarities arising at the contact line. And let's take you again. A USAID model gives alé...!
 Thank you very much for the introduction.  I apologize the long title.  I didn't know how to combine these two areas.  I guess most of you were asked themselves, like, why proteins now and probably don't  have any connection, academic connection to proteins, but I'm sure you maybe already  consume them this morning at your breakfast.  With this I would like to introduce into the industrial relevance at the example of milk  home organization.  So milk is a multi-phase system which basically contains water, oil, and proteins.  And what happens in milk home organization, you force, you apply some stress, you force  it through membrane, for example, and then you create fine monodysperous emulsion.  And on the right hand side I would like to show you that there's a schematic plot of  the stress residence time.  So you can have a long residence time of those proteins and those droplets in the system  with a relatively low shear stress.  You can have medium exposure to shear stress and time.  And you can have long and short and high stress peaks.  So the residence time, the stress rem, and the level of stress are very important factors  for this kind of process.  And the question is then what is the influence on drop breakup itself and break up and protein  degradation.  The motivation comes from the premix membrane emulsion.  So you basically start with a rough premix.  It's already water, oil, and proteins.  You force it with a pressure gradient through a membrane to create some fine and monodysperous  emulsions.  And how this works is due to shear and strain stress inside the membrane.  And the hypothesis is that the protein absorption affects the wetability of those membranes  and which also affect then the break up mechanism.  I would like to look at two different scales today, the macro and micro scale, which is  the fluid dynamics, so the droplets version inside the membrane, the influence of the  wetability on the droplets version mechanism inside.  And on the other scale, the atomic scale with molecular dynamic simulations, the adsorption  of the protein as at the fluid fluid interface.  So it basically adopts to the fluid interface to reduce interfacial tension there and therefore  stabilize emulsions.  And also the adsorption to fluid solid interfaces as in experiments we have seen membrane clogging,  so protein adoption to the membrane itself.  To start, I would like to talk about the influence on the drop dispersion mechanism.  And we identified the wall region as a region of high shear stress, so we would like to  have a closer look at the wall interactions.  This is I accidentally got to the end.  Right, so the drop dispersion in forest media is a very complex phenomenon.  We have multiple semi-chains break up events.  We have a complex and very irregular structure.  And this leads to the fact that the break up mechanism inside of those membranes is pretty  much unknown.  What can we do to address this?  We can reduce complexity, of course.  We will look at the capillary break up, so we just have a single droplet in a single  pour and we can quantify therefore influencing factors on the single break up mechanism.  With the numerical setup, we use oil and water, so with the according fluid properties,  our simulation parameters, we vary the contact angle in our simulations.  The dimensions, we have a 200 micrometer pour, which is 6 millimeters long and we push a  droplet of 500 micrometers into this pour with very capillary numbers as low as 1e-3  to 1 with the according Weber-Andrejnod's numbers.  The mesh, we use a hexahedral mesh size over a mesh size of 12 micrometers and we refined  it down to 3 micrometers at the interface with an adaptive mesh refinement.  We keep a current Fridtisch-Lewis criteria as low as 0.5 to satisfy the advection of  the fluid phase within our domain and we also keep this time step as low as necessary to  resolve capillary waves to account for various velocities.  Further, we use open foam and we use a solver called Interflow, so it's basically a volume  of fluid approach.  You have a phase fraction based interface capturing.  This means you have a phase fraction that denotes the fluids.  For example, one here would be the fluid A, zero would be the fluid B and in between 0.5  is where our interface is located.  We solved the Navistock's equation with continuity and momentum equation.  We applied for surface tension a continuum surface force model from Rekkel and Korten,  but recently we also implemented a sharp surface tension force as these systems are  so small that they are really affected by spurious velocities which occur from discretization  errors and also errors from the implementation of the surface tension force.  To account for the wall interaction, we use a dynamic contact angle model which includes  the function of Kistler which basically tells us that if we increase the velocity, our contact  angle will also increase.  We use a full three-dimensional hexahedral mesh with interface refinement around 0.5.  Here I would like to show you some examples of four different simulations from hydrophilic  to hydrophobic membranes.  We can first divide this into five different regions.  We have a leading front of the polymers droplet.  We have a leading contact line and we have a wetting region and a trailing contact line  with the overall trailing side.  We now only concentrate on the leading front.  We can see in the top right figure that the wave length of the leading front instability  is dependent on the wetability.  We conducted linear stability analysis for this from Lenz and Kumar to explain this.  This is basically a model which takes confined liquid film trial layers and calculates the  growth rate and the dominant wave number according to the layer height because we could observe  that for our hydrophobic membranes, our wall layer of continuous fluid was actually thicker  than for the hydrophilic ones.  The results only show that the growth rate is increasing but not the wave number is increasing.  The question still remains how does the wetability affect the unstable modes of the leading front  instability?  One explanation is we saw that the first moment the droplet enters the pore and it touches  the pore is really crucial part.  You might expect that if a droplet enters the membrane, it would directly attach to  the membrane itself.  So we have one located but actually with higher capillary numbers, it enters the pore, it  necks at the entrance and the interface advex to the wall at later deeper inside the pore.  You could see that this necking is also dependent on the contact angle.  So if the more hydrophobic the membrane is, so the better the oil wets the membrane, the  bigger the wetting.  And we could see that at point two, the interfacial advection to the wall has shown some radial  velocity which was also increasing with increasing wall wetting of the oil droplet.  The droplet now further emerges into the pore.  The continuous phase eventually gets trapped between contact lines.  And what happens then is we can explain it by some effect called capillary venturi.  So it's basically a venturi effect inside a capillary where capillary forces and hydrodynamic  forces stay in competition to each other.  Since they stay in competition to each other, they will be drawn inside, this volume will  be drawn inside of the capillary to the pore center, which on the right hand side you can  see that due to this entrapped phase and due to a contact line instability, this fingering  instability is formed on the right lower figure.  In easier words, if we see the enclosed entrapped continuous phase as a droplet, we have a contact  line velocity directly at the pore and we have a mean pore velocity inside the pore.  And the gradient leads to the fact that the water droplet travels inside the pore and  forms this water droplets in oil.  Here I would just like to give you some regimes of those fingering instabilities.  On the left hand side, we can see probably the most famous fingering instability, which  is the displacement of more viscous fluid by a less viscous fluid.  In the middle, we can see a fingering instability that's existing because of the receding unstable  interface, so contact line instability.  And on the right hand side, we can see a droplet dropped on the surface with a high impact  velocity and from this rim surrounding the droplet, we can see fingers emerging.  This to the fluid dynamics, now I would like to continue to the atomic scale with the molecular  dynamics just to keep in mind, we look at the absorption of protein at the fluid fluid  interface for the reduction of interfacial tension and therefore stabilizing the droplets.  On the other hand, we look at absorption to fluid solid interfaces, so the membrane clogging  we observed in experiments.  I would like to look at the PASA immobilization and relate the experimental literature to it.  Absorb PASA to Zilliqa has shown an increased activity and also has shown a specificity  and an anchor selectivity.  They count some lid residues of this protein for this modulation of the activity and the  specificity.  What do I mean with lid residues?  So the PASA is not only protein, it's also an enzyme, so it has some functional groups  and it has a lid which opens the functional groups and closes the functional groups.  Our numerical domain looks something like this, so we have a bulk of water, we have our protein,  we have an interface which is silica or oil, no case, and we have water again, because we  are dealing with periodic boundary conditions.  We have to equilibrate our system and some preliminary research we have to do as rotational  analysis.  The PASA is quite a chunky protein and we cannot simply cannot wait for it to be attracted  by the surface itself, so we have to do some preliminary research in order to account for  this issue.  What we do is we put the protein, place it in proximity to the interface, then we run  a static MD run where we just calculate one molecular dynamic step, we save the interaction  forces between the protein and the interface, then we rotate it, keeping the distance constant  to the interface, run the MD run, save the interaction forces, and what we then have  is basically a sampling of the surface and the interaction potential of the protein to  the interface and we end up with the most attractive position of the protein relative  to the interface from which we can run dynamic simulations then.  Just a quick estimate for 500 nanoseconds, we need like 84 hours on a 1000 CPU cores,  so we cannot really reach time scales, we can reach in fluid dynamics and this I will  also address shortly.  So I already talked about activation and increased activation due to adsorption, here I want  to further clarify this fact on the left hand side, we can see the passive or inactive  depasal conformation, I highlighted the lid residues in purple and if you compare from  left to right you can see that these lid residues are in a different position, they expose on  the right hand side the active center which is basically the place where the catalytic  reaction happens inside the enzyme and they expose this part in the active conformation  which then allows ligands actually to dock inside of this enzyme and for the enzyme to  catalyze the reaction.  We put our protein on surfaces, two different surfaces, in oil and on silica and what you  can see here from the snapshot is basically that we have different adsorption configurations  at liquid-liquid and liquid-solid interfaces, at liquid-liquid interfaces it adsorbs with  the lid residues facing into the oil phase whereas on the right hand side the silica  it exposes the lid residues to the water phase, I remove the water here because otherwise  you wouldn't see anything, so just in case you're wondering.  Excessibility of the active center and the lid residues for ligands on the silica adsorption.  We look now further into the silica adsorption, we want to know what exactly happens, we could  just see that it adsorbs so we can see by plotting the lid residues, each individual lid residues  I plotted the distance between and on this graph we can see that at around 250 nanoseconds  it starts to move and it starts to deviate from its equilibrium position which can be  also confirmed by just looking at snapshots and highlighting the lid residues we can see  it moves further to the active center.  The question is what triggers the movement, we remember that the lid residues were physically  distanced from the interface so there has to be something that triggers this movement  close to the interface itself.  For this we did some binding analysis and we basically took every residue of this protein  and measured the distance between the interface and the residue and whenever it was in a distance  where it could possibly build hydrogen bonds we marked this as binding and what you end  up with is this kind of graph so you can see from this that you have constant binders which  are basically constantly attached to the surface and frequent binders then would look more  like this barcode structure.  Interestingly we can see that the residue 522 lifts at around 250 nanoseconds from the  surface which was previously a constant binder and which the result is the lid movement.  We did some reference simulations for this so we just simulated the protein just in bulk  water and we couldn't observe the lid's movement in these simulations.  Further we did this also for the passive starting structure and there plotting the distance of  the individual lid residues we couldn't see any lid movement whatsoever.  So the question still remains is the protein now active or inactive?  When we speak of interfacial activation or not and how can we address and determine protein  activity numerically?  First accessibility of the active center has to be guaranteed for a ligand actually to  dock inside of this protein and second of all the ligand has to bind inside so the catalytic  triad has to be still in a functional arrangement where it could possibly cut our ligand in  pieces.  To address the first issue we did some tunnel calculations with a tool called CAVA so what  you basically do is you go from the surface of the protein and find tunnels which are  connected from the outer surface to the catalytic triad and we can see on the left on the starting  configuration that we have an open access to the active center and in the let's say natural  case the ligands would just directly dock onto the protein and not even entering fully.  On the right hand side we can see a complex tunnel network which has developed which is  introduced by the bulk we confirm this in bulk simulations so the water introduces this  tunnel but we can say that the tunnels are stabilized due to the energy minimization  due to adsorption to this interface.  Still active or inactive?  Here I would like to show the catalytic triad what's the only thing you have to know is  that it works together so it has to be in a functional arrangement of an acid base in  a nucleophile and what happens is that a nucleophile in our case 0209 attacks our ligand with a  nucleophilic attack to cut it.  In our case we simulated a medium change triglyceride to address the questions if the catalytic triad  is still in functional arrangement and how does it actually dock inside?  This would in some give us an indication of the activity of the protein.  We did this for a C8 triglyceride on the left hand side you can see that it docks into the  starting configuration of the active protein and on the middle part you can see here that  0209 which is our nucleophile and our catalytic triad stabilizes this part of the ligand with  hydrophobic interactions whereas for the adsorb structure we couldn't observe these  interactions anymore so which would then give us an indication of the selectivity already  of this protein.  You varied the input structure, the docking structure and we did this with a C8, C6 and  C10 chain and for the starting configuration we couldn't we could see that it was docking  inside the protein but it was actually not interacting with the catalytic triad so it  was just stabilizing this arrangement but not actually in a position where it could possibly  catalyze the reaction.  Whereas on the right hand side for the adsorb structure we could see that the C10 chain  here is stabilized by our nucleophile.  So in sum we have an indication of the selectivity and an ancilloselectivity and ancilloselectivity  I forgot so we also put the enantiomere of this ligand inside the protein and docked  it there and we also couldn't observe interactions with the adsorb structure and our ligand.  Still this is a work in progress these are just some few examples which we have docked  inside we are running a lot more in order to really tell what is this adsorb structure  selective to and how can we determine for example which chain the experimentalists have  to make longer or shorter in order for the adsorb structure to accept it or not.  This I would like to come to the conclusion and to the outlook so the key findings from  the computational fluid dynamics were that wetability affects the most unstable mode  of the leading front instability that we have fingering in stability as a result of entrap  continuous phase from molecular dynamics we could see that we have first of all different  adsorption configurations of the parser liquid liquid and liquid solid interfaces.  We have seen that adsorption of the parser to SU2 interfaces leads to an accessibility  of the active center through a tunnel network which we could also imagine that we have an  improved removal of the catalytic product through the tunnel network and our ligand binding  simulations indicate different confirmations of the catalytic triad or the binding pockets.  The key questions are how can we combine CFD and MD results as I said I agree pretty limited  in the time space in the molecular dynamics and how can we combine these two things together  we can quantify shear stress for example in computation fluid dynamics and then apply  this shear stress to the molecular dynamics now that we found some stable configurations  where our proteins adsorb where we can determine if it's active or inactive we can actually  try now to destroy it in order to see to draw the whole picture what is the effect of forcing  it with a pressure gradient through a membrane maybe we destroy the proteins the adsorpt structures  maybe we don't and the last question is how does the adsorpt protein to liquid liquid  liquid solid and liquid liquid solid as we have seen it could be possible that it adsorbs  to to a solid liquid interface but then has still the the part exposed where it could  adsorb to a fluid fluid interface so how does this influence properties like what ability  in our use case this I would like to acknowledge a few people we also work for the molecular  dynamics part with the hybrid materials and interfaces group of professor Colin B. Chuckley  and yeah HLRN I would thank for computational resources and all of you for your attention  and open for questions.  Did you employ a special approaches for your protein folding also standard and the second  one is have how about the force feed validation for interacting of the protein with the surfaces  can you say how this was done in your force field model.  So you mean validation from the interaction process is it standard.  This is experimentally validated by the hybrid interface group of agreement so there is if  you're interested in publication about the experimental work and the interaction forces  between proteins and interfaces.  In a similar direction but kind of potency use for the MD because you have different  materials proteins and unorganic materials on your simulation.  But for the unorganic materials as I said is done by the group by the hybrid materials  and interfaces group they did some experiments and parameterized the force fields for it.  And for the oil we can just use also slightly changed standard direction potentials.  So for the MD part did you only look at the structural properties or did you consider  something like a chart densities as well.  Yes we did but I'm just starting to look into this first we were just focusing on the structures.  Okay and for the interaction parts.  Did you consider something like QMM approaches as well.  Okay thanks.  The contact and your model.  Given the parameter or is it an unknown problem.  So it's given parameter by the material we use but it's a dynamic in terms of velocity  dependence so as the velocity increases and this is basically what the Kisla model says  is that at the capillary number of one our contact angle would always increase towards  180 degrees.  So what it does is it basically takes the velocity at the contact line and then calculates  the capillary number there in order to change the direction of the surface force applied  there to apply the contact angle.  What is your boundary condition in the next two to the contact point?  No slip boundary condition.  Right now yes for this it was no slip but we currently implementing another slip condition  in order to account for the non-integrable stress similarities arising at the contact  line.  And let's take you again.  A USAID model gives alé...!
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Thank you very much for the introduction. I apologize the long title. I didn't know how to combine these two areas. I guess most of you were asked themselves, like, why proteins now and probably don't have any connection, academic connection to proteins, but I'm sure you maybe already consume them this morning at your breakfast. With this I would like to introduce into the industrial relevance at the example of milk home organization. So milk is a multi-phase system which basically contains water, oil, and proteins. And what happens in milk home organization, you force, you apply some stress, you force it through membrane, for example, and then you create fine monodysperous emulsion. And on the right hand side I would like to show you that there's a schematic plot of the stress residence time. So you can have a long residence time of those proteins and those droplets in the system with a relatively low shear stress. You can have medium exposure to shear stress and time. And you can have long and short and high stress peaks. So the residence time, the stress rem, and the level of stress are very important factors for this kind of process. And the question is then what is the influence on drop breakup itself and break up and protein degradation. The motivation comes from the premix membrane emulsion. So you basically start with a rough premix. It's already water, oil, and proteins. You force it with a pressure gradient through a membrane to create some fine and monodysperous emulsions. And how this works is due to shear and strain stress inside the membrane. And the hypothesis is that the protein absorption affects the wetability of those membranes and which also affect then the break up mechanism. I would like to look at two different scales today, the macro and micro scale, which is the fluid dynamics, so the droplets version inside the membrane, the influence of the wetability on the droplets version mechanism inside. And on the other scale, the atomic scale with molecular dynamic simulations, the adsorption of the protein as at the fluid fluid interface. So it basically adopts to the fluid interface to reduce interfacial tension there and therefore stabilize emulsions. And also the adsorption to fluid solid interfaces as in experiments we have seen membrane clogging, so protein adoption to the membrane itself. To start, I would like to talk about the influence on the drop dispersion mechanism. And we identified the wall region as a region of high shear stress, so we would like to have a closer look at the wall interactions. This is I accidentally got to the end. Right, so the drop dispersion in forest media is a very complex phenomenon. We have multiple semi-chains break up events. We have a complex and very irregular structure. And this leads to the fact that the break up mechanism inside of those membranes is pretty much unknown. What can we do to address this? We can reduce complexity, of course. We will look at the capillary break up, so we just have a single droplet in a single pour and we can quantify therefore influencing factors on the single break up mechanism. With the numerical setup, we use oil and water, so with the according fluid properties, our simulation parameters, we vary the contact angle in our simulations. The dimensions, we have a 200 micrometer pour, which is 6 millimeters long and we push a droplet of 500 micrometers into this pour with very capillary numbers as low as 1e-3 to 1 with the according Weber-Andrejnod's numbers. The mesh, we use a hexahedral mesh size over a mesh size of 12 micrometers and we refined it down to 3 micrometers at the interface with an adaptive mesh refinement. We keep a current Fridtisch-Lewis criteria as low as 0.5 to satisfy the advection of the fluid phase within our domain and we also keep this time step as low as necessary to resolve capillary waves to account for various velocities. Further, we use open foam and we use a solver called Interflow, so it's basically a volume of fluid approach. You have a phase fraction based interface capturing. This means you have a phase fraction that denotes the fluids. For example, one here would be the fluid A, zero would be the fluid B and in between 0.5 is where our interface is located. We solved the Navistock's equation with continuity and momentum equation. We applied for surface tension a continuum surface force model from Rekkel and Korten, but recently we also implemented a sharp surface tension force as these systems are so small that they are really affected by spurious velocities which occur from discretization errors and also errors from the implementation of the surface tension force. To account for the wall interaction, we use a dynamic contact angle model which includes the function of Kistler which basically tells us that if we increase the velocity, our contact angle will also increase. We use a full three-dimensional hexahedral mesh with interface refinement around 0.5. Here I would like to show you some examples of four different simulations from hydrophilic to hydrophobic membranes. We can first divide this into five different regions. We have a leading front of the polymers droplet. We have a leading contact line and we have a wetting region and a trailing contact line with the overall trailing side. We now only concentrate on the leading front. We can see in the top right figure that the wave length of the leading front instability is dependent on the wetability. We conducted linear stability analysis for this from Lenz and Kumar to explain this. This is basically a model which takes confined liquid film trial layers and calculates the growth rate and the dominant wave number according to the layer height because we could observe that for our hydrophobic membranes, our wall layer of continuous fluid was actually thicker than for the hydrophilic ones. The results only show that the growth rate is increasing but not the wave number is increasing. The question still remains how does the wetability affect the unstable modes of the leading front instability? One explanation is we saw that the first moment the droplet enters the pore and it touches the pore is really crucial part. You might expect that if a droplet enters the membrane, it would directly attach to the membrane itself. So we have one located but actually with higher capillary numbers, it enters the pore, it necks at the entrance and the interface advex to the wall at later deeper inside the pore. You could see that this necking is also dependent on the contact angle. So if the more hydrophobic the membrane is, so the better the oil wets the membrane, the bigger the wetting. And we could see that at point two, the interfacial advection to the wall has shown some radial velocity which was also increasing with increasing wall wetting of the oil droplet. The droplet now further emerges into the pore. The continuous phase eventually gets trapped between contact lines. And what happens then is we can explain it by some effect called capillary venturi. So it's basically a venturi effect inside a capillary where capillary forces and hydrodynamic forces stay in competition to each other. Since they stay in competition to each other, they will be drawn inside, this volume will be drawn inside of the capillary to the pore center, which on the right hand side you can see that due to this entrapped phase and due to a contact line instability, this fingering instability is formed on the right lower figure. In easier words, if we see the enclosed entrapped continuous phase as a droplet, we have a contact line velocity directly at the pore and we have a mean pore velocity inside the pore. And the gradient leads to the fact that the water droplet travels inside the pore and forms this water droplets in oil. Here I would just like to give you some regimes of those fingering instabilities. On the left hand side, we can see probably the most famous fingering instability, which is the displacement of more viscous fluid by a less viscous fluid. In the middle, we can see a fingering instability that's existing because of the receding unstable interface, so contact line instability. And on the right hand side, we can see a droplet dropped on the surface with a high impact velocity and from this rim surrounding the droplet, we can see fingers emerging. This to the fluid dynamics, now I would like to continue to the atomic scale with the molecular dynamics just to keep in mind, we look at the absorption of protein at the fluid fluid interface for the reduction of interfacial tension and therefore stabilizing the droplets. On the other hand, we look at absorption to fluid solid interfaces, so the membrane clogging we observed in experiments. I would like to look at the PASA immobilization and relate the experimental literature to it. Absorb PASA to Zilliqa has shown an increased activity and also has shown a specificity and an anchor selectivity. They count some lid residues of this protein for this modulation of the activity and the specificity. What do I mean with lid residues? So the PASA is not only protein, it's also an enzyme, so it has some functional groups and it has a lid which opens the functional groups and closes the functional groups. Our numerical domain looks something like this, so we have a bulk of water, we have our protein, we have an interface which is silica or oil, no case, and we have water again, because we are dealing with periodic boundary conditions. We have to equilibrate our system and some preliminary research we have to do as rotational analysis. The PASA is quite a chunky protein and we cannot simply cannot wait for it to be attracted by the surface itself, so we have to do some preliminary research in order to account for this issue. What we do is we put the protein, place it in proximity to the interface, then we run a static MD run where we just calculate one molecular dynamic step, we save the interaction forces between the protein and the interface, then we rotate it, keeping the distance constant to the interface, run the MD run, save the interaction forces, and what we then have is basically a sampling of the surface and the interaction potential of the protein to the interface and we end up with the most attractive position of the protein relative to the interface from which we can run dynamic simulations then. Just a quick estimate for 500 nanoseconds, we need like 84 hours on a 1000 CPU cores, so we cannot really reach time scales, we can reach in fluid dynamics and this I will also address shortly. So I already talked about activation and increased activation due to adsorption, here I want to further clarify this fact on the left hand side, we can see the passive or inactive depasal conformation, I highlighted the lid residues in purple and if you compare from left to right you can see that these lid residues are in a different position, they expose on the right hand side the active center which is basically the place where the catalytic reaction happens inside the enzyme and they expose this part in the active conformation which then allows ligands actually to dock inside of this enzyme and for the enzyme to catalyze the reaction. We put our protein on surfaces, two different surfaces, in oil and on silica and what you can see here from the snapshot is basically that we have different adsorption configurations at liquid-liquid and liquid-solid interfaces, at liquid-liquid interfaces it adsorbs with the lid residues facing into the oil phase whereas on the right hand side the silica it exposes the lid residues to the water phase, I remove the water here because otherwise you wouldn't see anything, so just in case you're wondering. Excessibility of the active center and the lid residues for ligands on the silica adsorption. We look now further into the silica adsorption, we want to know what exactly happens, we could just see that it adsorbs so we can see by plotting the lid residues, each individual lid residues I plotted the distance between and on this graph we can see that at around 250 nanoseconds it starts to move and it starts to deviate from its equilibrium position which can be also confirmed by just looking at snapshots and highlighting the lid residues we can see it moves further to the active center. The question is what triggers the movement, we remember that the lid residues were physically distanced from the interface so there has to be something that triggers this movement close to the interface itself. For this we did some binding analysis and we basically took every residue of this protein and measured the distance between the interface and the residue and whenever it was in a distance where it could possibly build hydrogen bonds we marked this as binding and what you end up with is this kind of graph so you can see from this that you have constant binders which are basically constantly attached to the surface and frequent binders then would look more like this barcode structure. Interestingly we can see that the residue 522 lifts at around 250 nanoseconds from the surface which was previously a constant binder and which the result is the lid movement. We did some reference simulations for this so we just simulated the protein just in bulk water and we couldn't observe the lid's movement in these simulations. Further we did this also for the passive starting structure and there plotting the distance of the individual lid residues we couldn't see any lid movement whatsoever. So the question still remains is the protein now active or inactive? When we speak of interfacial activation or not and how can we address and determine protein activity numerically? First accessibility of the active center has to be guaranteed for a ligand actually to dock inside of this protein and second of all the ligand has to bind inside so the catalytic triad has to be still in a functional arrangement where it could possibly cut our ligand in pieces. To address the first issue we did some tunnel calculations with a tool called CAVA so what you basically do is you go from the surface of the protein and find tunnels which are connected from the outer surface to the catalytic triad and we can see on the left on the starting configuration that we have an open access to the active center and in the let's say natural case the ligands would just directly dock onto the protein and not even entering fully. On the right hand side we can see a complex tunnel network which has developed which is introduced by the bulk we confirm this in bulk simulations so the water introduces this tunnel but we can say that the tunnels are stabilized due to the energy minimization due to adsorption to this interface. Still active or inactive? Here I would like to show the catalytic triad what's the only thing you have to know is that it works together so it has to be in a functional arrangement of an acid base in a nucleophile and what happens is that a nucleophile in our case 0209 attacks our ligand with a nucleophilic attack to cut it. In our case we simulated a medium change triglyceride to address the questions if the catalytic triad is still in functional arrangement and how does it actually dock inside? This would in some give us an indication of the activity of the protein. We did this for a C8 triglyceride on the left hand side you can see that it docks into the starting configuration of the active protein and on the middle part you can see here that 0209 which is our nucleophile and our catalytic triad stabilizes this part of the ligand with hydrophobic interactions whereas for the adsorb structure we couldn't observe these interactions anymore so which would then give us an indication of the selectivity already of this protein. You varied the input structure, the docking structure and we did this with a C8, C6 and C10 chain and for the starting configuration we couldn't we could see that it was docking inside the protein but it was actually not interacting with the catalytic triad so it was just stabilizing this arrangement but not actually in a position where it could possibly catalyze the reaction. Whereas on the right hand side for the adsorb structure we could see that the C10 chain here is stabilized by our nucleophile. So in sum we have an indication of the selectivity and an ancilloselectivity and ancilloselectivity I forgot so we also put the enantiomere of this ligand inside the protein and docked it there and we also couldn't observe interactions with the adsorb structure and our ligand. Still this is a work in progress these are just some few examples which we have docked inside we are running a lot more in order to really tell what is this adsorb structure selective to and how can we determine for example which chain the experimentalists have to make longer or shorter in order for the adsorb structure to accept it or not. This I would like to come to the conclusion and to the outlook so the key findings from the computational fluid dynamics were that wetability affects the most unstable mode of the leading front instability that we have fingering in stability as a result of entrap continuous phase from molecular dynamics we could see that we have first of all different adsorption configurations of the parser liquid liquid and liquid solid interfaces. We have seen that adsorption of the parser to SU2 interfaces leads to an accessibility of the active center through a tunnel network which we could also imagine that we have an improved removal of the catalytic product through the tunnel network and our ligand binding simulations indicate different confirmations of the catalytic triad or the binding pockets. The key questions are how can we combine CFD and MD results as I said I agree pretty limited in the time space in the molecular dynamics and how can we combine these two things together we can quantify shear stress for example in computation fluid dynamics and then apply this shear stress to the molecular dynamics now that we found some stable configurations where our proteins adsorb where we can determine if it's active or inactive we can actually try now to destroy it in order to see to draw the whole picture what is the effect of forcing it with a pressure gradient through a membrane maybe we destroy the proteins the adsorpt structures maybe we don't and the last question is how does the adsorpt protein to liquid liquid liquid solid and liquid liquid solid as we have seen it could be possible that it adsorbs to to a solid liquid interface but then has still the the part exposed where it could adsorb to a fluid fluid interface so how does this influence properties like what ability in our use case this I would like to acknowledge a few people we also work for the molecular dynamics part with the hybrid materials and interfaces group of professor Colin B. Chuckley and yeah HLRN I would thank for computational resources and all of you for your attention and open for questions. Did you employ a special approaches for your protein folding also standard and the second one is have how about the force feed validation for interacting of the protein with the surfaces can you say how this was done in your force field model. So you mean validation from the interaction process is it standard. This is experimentally validated by the hybrid interface group of agreement so there is if you're interested in publication about the experimental work and the interaction forces between proteins and interfaces. In a similar direction but kind of potency use for the MD because you have different materials proteins and unorganic materials on your simulation. But for the unorganic materials as I said is done by the group by the hybrid materials and interfaces group they did some experiments and parameterized the force fields for it. And for the oil we can just use also slightly changed standard direction potentials. So for the MD part did you only look at the structural properties or did you consider something like a chart densities as well. Yes we did but I'm just starting to look into this first we were just focusing on the structures. Okay and for the interaction parts. Did you consider something like QMM approaches as well. Okay thanks. The contact and your model. Given the parameter or is it an unknown problem. So it's given parameter by the material we use but it's a dynamic in terms of velocity dependence so as the velocity increases and this is basically what the Kisla model says is that at the capillary number of one our contact angle would always increase towards 180 degrees. So what it does is it basically takes the velocity at the contact line and then calculates the capillary number there in order to change the direction of the surface force applied there to apply the contact angle. What is your boundary condition in the next two to the contact point? No slip boundary condition. Right now yes for this it was no slip but we currently implementing another slip condition in order to account for the non-integrable stress similarities arising at the contact line. And let's take you again. A USAID model gives alé...!
time of those proteins and those droplets in the membrane emulsion. So you basically start with a with molecular dynamic simulations, the adsorption to fluid solid interfaces as in on the drop dispersion mechanism. And we according fluid properties, our simulation different simulations from hydrophilic to results only show that the growth rate is increasing but not the wave number is increasing. increasing wall wetting of the oil droplet. The droplets in oil. Here I would just like to give interface for the reduction of interfacial tension and therefore stabilizing the droplets. solid interfaces, so the membrane clogging we residues? So the PASA is not only protein, it's constant binders which are basically constantly simulations. Further we did this also for the stabilized due to the energy minimization due to that it works together so it has to be in a could adsorb to a fluid fluid interface so how a few people we also work for the molecular between proteins and interfaces. In a similar we were just focusing on the structures. Okay and something like QMM approaches as well. Okay parameter or is it an unknown problem. So it's order to change the direction of the surface