Okay, so rather than, you adding all these components you already know which G it is, right? So you use a bacteria to only express, and purify, the synaptobrevin protein. You just purified this only protein, okay? And then in a reconstitute test tube system, you just only added that protease. Seeing that the protease, the toxin, itself, will directly cleave this synaptobrevin into this molecular. And as if it does, because you already identified the cleavage yourself. Now you can mutate that cleavage site, right? And the thing that, because again, the protease are very specific. It just do not randomly cleave anything. It needs to have a high signal specificity, so it can just mutate to that site to see whether it will cleave or not, okay? And they didn't do that. But if they do it, they found, the toxin will cleave that and mutating the egg sack will completely eliminate that. Then, what else are you going to do? Do you have direct evidence it is this cleaviging? That blocked transmitter release. Well, this is just correlated. You add this toxin in your [INAUDIBLE] and this has been cleaved, okay? Maybe there's something else that is on cleaved, because here it's just too dirty, right? You want to suggest a huge number of protein. You probably couldn't see it, even if that's clearly there, right? So, you don't have any evidence to eliminate. Maybe there's additional components that have been cleaved, right? And therefore, do you have direct evidence that this cleaveging, is a function of significance for transmittal release? You don't, you only saying, adding this protease, this be cleaved. And adding a protease, there's no transmitter release. But if you know your [INAUDIBLE] the image itself is important to block transmitter release, right?. How are you going to do it? How are you going [INAUDIBLE] experiment prove that? So one way you can do is, well, you already identified this is very critical, right? So if you knocking out, so right now you just clearly, it does not have transmitter release. If you have a way to completely eliminate it, knocking out, by genetics, what's the prediction? Probably no transmitter release, or you cut it into half. There's no transmitter release. Now you eliminate, probably there's no transmitter release, right? How are you going to do it? Well, maybe you can knocking out. But think about if it's so important for the transmitter release, you are creating a complete knockout. Maybe it's a dead mouse, right? That mouse cannot breathe, do not have a heartbeat. So if we are going to do it, you can do a conditional no only in the adulthood that eliminate this protein, right? And if we want to demonstrate that this is indeed very important, you can put him back. Homogeneity already mutates this sequence that prevent this cleavage, right? So you can rescue this no call with this protease insensitive mutant. And if that can rescue, and then with that, can rescue mouse, you can fully add this toxin. Your prediction will be that since the toxin cannot cleave it, right? Even if the toxin might be able to cleave other dirty things, that you could not identify, if you add that toxin, there's no further effect, why you can get a very strong conclusion. Functionally, this toxin only recognizes this protein, because if I only rescue with the mutant to prevent cleavage of these other proteins, this only protein, it doesn't matter. Maybe this toxin will cleave other dirty components. It has no effect on transmitter release, right? So with that, you can combine with this toxicology. And with molecular biology, with this genetics, to bulletproof demonstrating that a specific molecular component is a key component, that regulate in transmitter release, okay? And we are going to discuss more about how to further identify the molecular components in that process. And one question for you guys to think before our next class is, after your proving using the no [INAUDIBLE] no key toxin rescue, blah, blah, blah, experiment. If you have done all those experiments, what other experiments you are going to do, to identify more molecular components? More important machineries that will control transmitter release? Okay, after all demonstrating the functional importance, what other approaches are you going to do? Knowing the fact of that and how are you going to do it? How are you going to design the experiment to getting out more components, and then identify their function? Okay? all right? So, let's just conclude with this figure for today, all right? And then we are going to continuing the discussion in the next class. And then I'm going to ask you guys, what will be your experiment, after knowing all those things, okay? So, last time we discussed the identification of one of the special toxin that can inhibit transmitter release. And we discussed the toxin. And by toxicology, or biochemical analysis, the Italian group, identify a molecule, a protein called synaptobrevin. Okay, which is label here, after being cleaved the transmitter reduce will be block, okay? And we were asking, how this identification of transmitter Release the substrate, could help us understand the molecular process? Controlling the whole process, the whole cascade of interactions that my regulating transmitter is, okay? Now here is the time that you guys can share with your wisdom. Well, they already identified synaptobrevin. So if you are the scientist with unlimited resources at that time, what will you do to unwrap the detailed molecular mechanism of the controller transmitter release process? Which will be essential for nerve communication for the understanding of neuronal communication, computation, and information processing. So, after identifying one component that controls the transmitter release, well, if you think there's additional components, right? And if they are working together, then most likely they might be in the same complex, right? So one way to identify it, is to identify which might be the components directly interact with the synaptobrevin. So you're already identifying the synaptobrevin, this protein, right? And previously, we already discussed or identified this protease. Some of you propose that using affinity purification. In principle, could work. But the problem for that protease, you will turn over, you will cut the substrate. And the substrate will be released, okay? So in that case, it will be difficult to directly affinity in reach the components. But in this case, if it's synaptobrevin, after identifying, the first thing you'll do will be, look at it's domain, look at it's structure, to see whether it has any unique domains that you can guess one maybe it's function, right? For example, if we had some calcium binding domain then you'll be aha, this will apply to calcium. Will there be a calcium sensor? Right, that will be one of the straight forces to test, okay? It will have other kind of domain. Well, maybe you will be [INAUDIBLE] if it has other interacting domains. If there is no more protein, well then your out of luck, because there's no proceeding information about this protein, okay? But for a protein, then the obvious thing to do is to identify is associate component, okay? And you know isolation will be one good way, okay? So you raise the antibody for this synaptobrevin. And then you pull that down. And then you look at what other component that you will be dragging down, okay? How do you know even, if after you're doing the immuno-isolation? How do you know which protein is which protein? You can just put out the whole compass. And then you run the SDS page out to separate the protein by their molecular weight. And you identify this three different molecule weight. How do you know which protein might be, they might be? Great, so this is the. As long as you identify the component, right? Then you can do the mass special analysis N-terminal Edman degradation. You can sequence the peptide sequence, okay? After you getting some peptide sequence, you search the database. You can identify which protein that accounts, which gene that accounts for this peptide sequence. This is the same way as we identified a synaptobrevin, right? Only after identified the binding components, then one needs to demonstrate these binding components. Whether they are functionally important or not, right? Okay, how do you demonstrate those binding components are functionally important or not? Well, genetically eliminate that by knocking out to see whether the transmitter release is auto or not, right? And then demonstrating that this is functionally important. Then one might try to worry about whether this is the A = B is 1 versus 5 relationship or 1 versus 10 relationship, okay? Without knowing the binding component, without knowing whether they are essential or not, who cares about their reaction ratio? Maybe it's just nonspecific binding, okay? And there indeed will be nonspecific binding inside the cell. There's so many different proteins and lipids that are floating around. Okay? So what might be other ways to identify the interaction? So, what will be other methods to identify to the protein might be associated with synaptobrevin? There are other methods that people can use, for example, use yeast two-hybrid, right? That, using a method that this is a chemically put down to identify the direct interactions. You can also use the yeast two-hybrid method. Again, for those of you who don't know what is yeast two-hybrid, please check. Basically, it's a way that you fill this protein, one protein bathed with transgression activation domain. And the other one was a DNA binding domain. And if these two are associated with each other, you would try some marked expression. Okay, so in that way you can try to identify the component. But the difficulty is, this synaptobrevin is a transmembrane protein, okay? So for the yeast two-hybrid, massive, it usually only works with the cytosolic protein because it requires the gene is present to identify the interactions, okay, all right? So indeed as you will see, some people have used. And we are going to discuss that. But, let me just ask you other questions. How do the synaptobrevin recognize exception? You already that the protease will recognize the synaptobrevin, okay? And, so how do they recognize it? Well there's studies that people using the crystal structure to study how the protease recognize the substrate, okay
