 So just to check that we're on the same wavelength, I'm going to do a small quiz for us. Me too. Here I have a plain simple helix with some residues there, green, blue. Here I have another plain simple helix boring just polyalanine. Which one of this will go into the membrane when I'm asking the translocon to process it? To make things easier, I'm going to add some charges to those residues. I wish I could do this in person with you, but hopefully you're all going to say that that's not likely to go into the membrane. And that's also what I would have guessed if I was just showing the sequence and didn't know the answer. And yet that is possibly the most important helix in your life. It's the reason you exist. This is part of a voltage-gated ion channels, and in particular the parts that senses the voltage. And those channels are, for instance, responsible for closing the egg once they're first realized. If this didn't happen, you wouldn't exist, and neither would I. It's also responsible for every heartbeat in our bodies. So it's kind of important. Why is that helix a membrane protein helix? Well, beats me, I almost said. This is the entire channel, and this is the helix. There are four copies of it, actually. You will probably hear more about these channels later. But why on earth is this charged? Well, this is a protein that should react in response to a voltage change, and that's an electric field change. It makes a lot of sounds to have a charge, because a charge in an electric field will feel a force. And if you now want to create a machine, getting it to move is the first step. That's, of course, why it helps create all these iron currents that, in turn, leads to nerve impulses and everything. And yet we don't know about that molecular mechanism and how it happens. The first person to realize the importance of the voltage sensor itself, I would argue, is Yasushi Okamura. So Yasushi is a Japanese researcher. He realized that the same type of helices occur in a few small organisms. CO9 test analysis, one of them. But in these organisms, he didn't only see a full voltage gate, the voltage gated ion channel. He saw a small voltage sensor only protein. That is a protein that consisted only of those four helices forming the voltage sensor. Occasionally, they occur in pairs and everything, but that's not so important. But the point is that the large tetrameria I showed you, they had six helices times four. But it turns out that we can cut off just the four first helices of the voltage sensor and it still senses voltage and can conduct EG protons. There is also a peculiar pattern here that the first helix is hydrophobic. Second is, sorry, first helix is hydrophobic. Second is hydrophobic. The third helix, well, maybe halfway. But it goes in because its peers are already there. The fourth helix, definitely not. It's even hydrophilic with all those charges. So somehow the previous three helices help stabilize the fourth helix. I think I have an illustration of the structure of it. Yes, I do. The helix here in the rear, the blue one, is the one that has all those arginine side chains that is responsible for somehow moving up and down. And people fought a decade about this before we eventually converged on models. So one way or another, they will have to change the structure to open the central ion channel. There are a few different models, but we and some others believe that the helix should move roughly straight up and down, maybe rotate a bit. There were people who assumed that there should be a paddle moving straight through the membrane. And there have also been ideas that maybe you just had some sort of tilting mechanism that you don't really move up and down, but you're exposed alternatively either to the inside or the outside. I would argue that we won and the people eventually agreed on our model, but I bet other people will disagree with me. How do we know that that's true? Well, the idea here is that if it's moving that way, those positively charged residues should be stabilized by the lipids, right? That's why it would not work with negatively charged residues because there are much fewer positive charges deeper buried in the membrane. Rod McKinnon came up with a very smart way of testing this. So they took this voltage sensor and then they expressed it in a special membrane that was not the normal mix of all this Twitter ionic lipids, but that you had in particular only a dotap lipid. It's a bit strange lipid with only a single positive charge. And this simple, plain proteins that worked great when expressed in PC head groups, if you just had them in dotap head groups, the protein doesn't work. It can't open. Now, you could argue that might be due to the lipid complete misfolding or something, but the neat test is that if you then just titrate and add a few drops of PC normal head groups to it, it starts working again. So it's the presence of negative charges fairly far down in the lipids that helps stabilize this voltage sensor either in the open upstate or in the closed downstate, which is a remarkably cool biological process. We know much more about this today. People have been able to do neutroscattering studies, molecular dynamics simulations. And in principle, I would argue that we have a pretty darn good consensus about the closed downstate two that we would normally only see when there is a polarized membrane. And you can't really get that in a crystal. We've also have good models for what happens when we say mutate this ratio. We know that this is all electrostatics. So in theory, if I start to alter some loops in the protein, well, if I introduce or remove charges there, I'm going to change the local electrostatic environment. And that means that it's going to be either easier or harder to move my four charges up or down. And that makes sense. If I measure this in electrophysiology, systematically, it bears out exactly where I place these charges. So it's a plain simple electrostatic problem, and yet one of the physiologically most important processes for us. Let's have a second look at what they learned. You already saw this one. Let's look at it from the top. Do you see this helix with the arginines exposed here? Now, you could say that in a real vaulted gaited ion channel, you're going to have the pore domain and stuff here. So this won't really be exposed to water. But remember, Yasocio-Kamura's result that they can exist in isolation, just these one, two, three, four helices. And in that case, you would expose the side chance here to the protein. There is no way that can happen. Sorry, the side chance here to the lipids. There is no way that can happen. The way we solve that is exactly the lipid head groups. So here, the helix is in its upstate. And in the upstate, these two residues up here, in particular, they have to form hydrogen bonds or salt bridges even to lipid carbonyls or even the phosphate groups in the lipids. But that's going to make them quite happy. Maybe the lipids have to stick down a little bit, but they don't have to interact with hydrophobic residues. Essentially, they snort a lot of it. These two other residues here in the middle, they are facing the red residues here, that is aspartic acid. So they form salt bridges and are quite happy. If we now move this entire helix down, they trade places. These residues that were previously out here, they're now going to face the red ones. While the residues that were previously facing the red ones, they are now pointing down and interacting with lipid head groups on the other side. It's a remarkably beautiful example of how lipids stabilize membrane proteins and membrane proteins get their function by moving between different states and using that.