 My name is Thea Lugo-Milla. I work at AU. I have Milena Korredik and Jan Skopil as my supervisors. And together with them, I have been working on, yeah, the fast-forwarding, the case in my cell, basically. Which is going to be the topic of today. And the objective of the work was to investigate... One second. The objective of the work was to investigate the structural effects due to the dephosphorylation of the case in my cell. And I'm going to talk a little bit more about that in a second. However, the objective of the talk today is to see whether SACs can actually be employed to study subtle structural changes in the my cell as a consequence of dephosphorylation. So, to those of you who don't know the case in my cell, it's a proteinaceous colloidal structure in milk. And it's composed of casein proteins, Kappa beta, Alpha 1 and Alpha 2. Kappa casein is on the outside. It's shown in green in the figure. And it is hysterically stabilizing the casein in my cell, preventing it from growing it, growing undefinitely and to sort of aggregating with the casein in my cells. And it's held together by different interactions, electrostatic and hydrophobic interactions. And the electrostatic ones are due to, or in part due to the phosphorylations of the caseins. And they bind calcium, forming so-called calcium phosphate nanoclusters. The hydrophobic interactions happens because the caseins are rather hydrophobic molecules, so they stick together by, yeah, by hydrophobic interacting. And especially part of the beta casein, which is in the figure shown in blue, is thought to be anchored only through hydrophobic bonds and stabilizing presumed water channels within the casein in my cell. Because the casein in my cell is a rather hydrated structure. And since the caseins are rather hydrophobic, there has to be some way to sort of like stabilize and include all of this water. And here comes in the beta casein, presumably, stabilizing the so-called water channels. So the idea behind this dephosphorylation work was actually to try to see if we can answer some of these questions, such as, well, can we even have dephosphorylation in a micellar system? I mean, does the phosphatase, does it actually enter the micellar and start working inside of it? And if so, are we actually able to alter the chemistry of the caseins and also the internal structure? And if so, what does this actually tell us on the original casein micellar structure? And I'm just going to disclaim it right now. I don't have the answers to these questions. Yes. So don't expect that from this talk. However, we have chosen small angle x-ray study is scattering to study the micellar structure, because it has previously been shown proven actually to be a quite useful tool to study the total structure of the micellar. What is shown here is the graph is a typical scattering profile of a casein micellar. And we have these three main scattering features, the low-cube scattering plateau, which many people, most people actually agree that it's due to the overall scattering of the micellar. And then we have the intermediate region where there's like a small bump or shoulder, and then we have the more pronounced high-cube shoulder here. And the bars represent the cure ranges over which previous investigators have recorded the scattering of the casein micellar. So, in order to actually understand and interpret the scattering data, we have to have models, mathematical models that can describe what the features mean. I mean, what kind of physical structures are we actually seeing? Do we have in this solution or suspension? I mean, people have done it. I mean, there's a model by Bushu by Ingham, which is actually based on a previous model by Jensko Pillaven. However, the structure of the dephosphorylated casein micellar has not been looked at before by small angle x-ray scattering. Mainly dephosphorylation has been studied in monomeric systems, so where the caseins are soluble and free-floating. So, what did I do? I basically created this series of samples consisting of resuspended casein micelles in native and increasingly demineralized environments, thereby causing a gradual casein dissociation. So, I have skim milk, ultracentrifugitive separating the serum phase from the micellar phase, discarding the supernatants and the micelles, the pellets, where we suspended in various permeate media. So, creating four samples, MCN 100, MCN 50, MCN 0, and MCN 50 DTA. So, MCN 100, in here, the pellet was resuspended in 100% permeate, so basically the native environment of the micelles minus the wave regions. So, this represents more or less the most native like micelle. Then we had the MCN 50, which is resuspended in 50% water and 50% permeate, MCN 0, 100% water, and MCN 50 DTA was resuspended in 50% permeate, and 50% 18 millimolar EDTA solution. So, we have this series of samples, series of suspensions here, in which we have decreasing ionic strength. Then they were either treated with phosphatase, I have abbreviated calf intestinal alkaline phosphatase, yeah, cap, and then or untreated. And then the suspensions were analyzed chemically or compositionally by reverse phase HPLC and structurally by small angle extracellular. So, understand the system more in depth, we also separated the suspensions soluble phases by ultra centrifuging them a second time obtaining the supernatants, which represents whatever we have soluble. And they were also analyzed in the same way. So, first, let's take a look at the, at the supernatants slash the backgrounds. And, and this is the reverse phase chromatograms. And the upper one is the protein composition of the micellar suspension and we can see we have all of the casings we have three kappa casein peaks, alpha is two alpha is one beta and some trace amounts of weight proteins. And then, just due to the resuspension in these various media with various mineral concentrations, we obtain different degrees of casein dissociation. So we can see that in the more native like my cell, which is the MCN 100, we don't have much casein dissociation we have a little bit is the blue curve down here. Then, replacing half of the soluble minerals with water, we obtain a little bit more casein dissociation, and even a little bit more when we suspend and just, just water. And then when we treat the micelles with EDTA we just, we actually pulled out quite a lot of casing. So we looked at what structures we could observe with small angle x-ray scattering in the soluble phase. And that's what we're going to look at here. And, and what we see is that with increasing amounts of casein soluble in the soluble phase. They make different structures, they have different structure scattering patterns or scattering profiles, meaning that they actually form some kind of structures out there. And this actually highlights why it's so crucial to use the right kind of sample for background subtractions when looking at the structures that you actually want to see. So we use these samples for background subtraction when looking at our actual micelles. These are just the take home messages from this slide. Go on. And this is, this is the sex of the actual untreated micelles. The main difference that we can observe is happening here in the intermediate region, at least among the MCN 150 and zero, and also in the intensity of the high Q shoulder. The intensity of the EDTA treated micelle is lower for all Q values. And I think that's because we saw that more casein dissociates due to the treatment. And that casein contributes to the scattering of the, of the background. And, and I mean, I'm not going to go too much into these also what this structurally means. The fact is just I mean my point is just that we can actually observe changes, even with when we have treated the micelles very mildly, we can observe small changes in the intermediate region and in the high Q shoulder. So what happens then when we start the phosphorylating the micelles figure a you saw before figure C is the suspend the cap treated suspensions, and we can see that we're actually able to separate the phosphorylated species from the dephosphorylated species marked in red. And what I noticed from this is that the yellow curb, which is the MCN 50 DTA, that is prone to way more dephosphorylation I mean it contains more dephosphorylated species than the other than the other samples and the other micelles. And, and, I mean, I guess it makes sense right because we dissociated more casings. When treating it with the DTA and I assume that the free flowing casings are more susceptible to cap attack to phosphate attack compared to the to the micellar ones. And so it seems that that the micelle does seem to have a like a limiting the structure itself seems to have a limiting factor. And when we look at the soluble faces, the supernatants. And, well, we see that what was before soluble here in the untreated micelles. So this case must be present already when when we add the phosphatase that's no longer present. So, so some so the phosphatase must have must have active on whatever casein was out there. And what then has happened to this dephosphorylated casein. I don't know. Do they have made particles on their colloidal particles on their own that then are sedimentable or do they just merely precipitate or are they do they attach to the casein micelle surface of some in some way. I don't know. However they're gone. And then in the EDTA treated sample in the day EDTA treated soluble face. We can see that it's mainly the dephosphorylated species that are present in the soluble face. So the mineral environment must have some kind of effect on how these dephosphorylated species act. Or behave. And then we looked at the sacks of these captured micelles. And this is what we see. Again, the main difference in scaling that we observed is in these in the intermediate region here. And in the, in the high q shoulder. And in the EDTA free to sample it behaves differently, which is, as I probably do to to the EDTA treatment itself, which is another kind of treatment that we're introducing so in. So, so even here also with with phosphatase treatment, we're actually able to to detect changes. Yeah, as I said before, I need to actually interpret on on what kind of structural changes we're observing physically speaking. We need a model and we are working on such a model or you can scroll pillows and has been working on such a model, and he has actually been able to refine a casein micelle model. And by combining the scattering of the casein micelle over a much wider q range. So we have combined the scattering from static light scattering, representing the first bar here. And with the the sacks scattering of small and small angle extra scattering in a low q confirmation with the scattering from a high q confirmation. And all of this put together with all of this put together he has been able to refine the model and to actually better quantitize quantify. What are the internal changes that are happening. Yeah, I look very much forward to actually start applying this model to my data is we can start verifying what kind of changes we're seeing. So, conclusively, well, changing the chemical environment of the micelle is clear results in some kind in some degree of casein dissociation and these particles for may form some, some kind of structures that may also be observed by small angle extra scattering. And well, my data suggests that that sacks can be used to study these subtle changes occurring to the structure of the casein micelle. And that was it for me. Questions. Thank you to yes. Tommy asked how did you control the pH of the system to compare water with the milk salt solution. Can you say that again and slowly. Yeah. How did you control the pH of the system to compare your water with the milk salt solution. Actually, I didn't. I didn't exactly control it. I am. I made sure that whatever I added was at the right pH. So I guess I control it. I control it beforehand. Does that make sense. We have to ask Tommy, if it made sense to him. I think it's, I mean, my point is that is this milk salts are extremely complex mixture of citrate and calcium. You can change everything under the summer. I'm going to say that and then if you change if you diluted, you change everything and you can affect the pH and the association of the whole system. This said, I think it's a very nice studies. Can I can I help there. Yeah. First of all, these, these systems are not extremely diluted. They are basically the same kind of protein concentrations and milk. And, and so the buffering capacity of the protein is high enough to keep the pH constant. And also the dilutions that we make are very similar to the dilutions that are made during concentration of milk by filtration. Which is known as not affecting the structure, or at least people think they're not accepting the structure, but obviously, the fact that Teya has managed to, to really subtract the right backgrounds from the scattering tells you that is actually changing the structure. Yeah. So, you know, it's, it's, that's my point. I agree completely. Alessandro has a question here. I assume the low q shoulder corresponds to the radius of generation of the whole casting micelle. What about the high q shoulder. It is very small. Is it a soft micelle. So, according to the model that you're so has been working on, it's due to the scattering of the calcium phosphate nano clusters and surrounded by a protein protein shell. I don't know how much is it say here Milena but I guess I mean it, he has found out that it's like this ellipsoidal like a structure component internally with the calcium phosphate nano clusters, the more electron dense and then surrounded by a fairly more electron dense layer of protein compared to whatever is outside of that. Yeah, Teya has been managing to collect data at high queues and low queues, like at a much larger range so so young school has been managing to put more absolute values to the core shell structure of the calcium phosphate nano cluster in the bottom range. And so he is, he's supposed to be able to now measure the, the thickness of the layer of the protein around the calcium phosphate nano cluster. That's, that's his model that's why he's, he's managed now with that with her data because of the background subtraction to really come up to, to absolute values for the value for the model. Okay. Just quick way, where do you, how do you achieve such low noise in the high queue range. We have a good instrument. Where is this thing sorry I didn't catch it. We have a good instrument. It's the answer. Yes, it's a bit unbelievable no low noise to the high curie. Thank you. Yeah sure it'd be interesting to discuss the details of the model and of course season fits from when you and gets around or you. Yeah, you're gonna have to talk to him. Yeah, I mean, I'm just one thing I'm sure he wouldn't know this but I just want to. I'm sure you know this paper that Greg Smith and the learning we're both here that it's on the sticky spheres model and so on so which is probably relevant for the static light scattering part also. So, let's just see, look at the time. What one quick question. The pattern where you showed the lower intensity of the ETA sample that this is not just a contrast thing that the EGA lowers the difference in electron density between the solvent and the proteins. Well, how could if I subtract the right background. That that that that was still the intensity absolute intensity of your particles are relying on the contrast between solvent and and the particle itself and that that is not. So remember that she's got a lot of stuff in solution now. Yeah, I'm just asking her background, her background subtraction decreases the value and it has a lot of scattering from the background that has to be subtracted. And of course, still the absolute intensity so if the contrast has changed the absolute intensity will change so you can check with to check with you. Anyway, that's perfect timing thank you to you.