 So does this make sense as far? Then it's both good and bad. You've understood a lot of things, but you've also forgotten something. I've explained why we might have residues in the inside, how they are stable once they are there, and how they might be stabilized by other helices. But at some point, those helices will have to go in. Good, I will talk more about that too in membrane protein biogenesis. But just to give you the background here, they have been able to measure how much it actually costs to insert different helices in a biological environment. I won't bother you with the method, good, I will actually cover that. But the net outcome of that is that you can get a cost of inserting different side chains as a function of the side chain. This makes a lot of sense functionally. The hydrophobic ones are easier to insert. The hydrophilic ones are difficult to insert, as we would expect. But don't you remember that I said it might cost 15 to 20 kcal to insert the charge? Here the y-axis says 3.5. So something is off here, either in this prediction, or in the insertion, or in the membrane. Why? Well, remember how I stressed at the start of this lecture that a membrane is not a lipid bilayer. And this is a membrane, while those pure 20 kcal or so is valid for a purely hydrophobic environment in a lipid bilayer. That difference comes from the 25 or 30% or so of the membrane that is not a lipid bilayer, but that is proteins. So if there is one mental view you should have when I say membrane instead of lipid bilayer, it's this one. A real membrane looks roughly something like this. This is a fake system. Just as a model to try to understand how the interactions work, I've just placed helices so that 30% of the atoms are helix rather than being lipids. But you see how much stuff there is now in the center that is not really lipids. Sure, these helices might have hydrophobic side chains, but the backbone and everything here, they're not as hydrophobic as the lipids. So maybe when we're inserting these helices, helix by helix, previous helices in the membrane, either the ones that came before me in my sequence or simply other helices are already there, can help stabilize a charge residue. And that definitely appears to be the case. We and others have done simulations with that, looking just at the isolated side chain from Arginine, the Guanidinium ion. And we love to interact with individual ions and even at the outskirts of the translocal. This side chain will be quite happy to interact both with waters and in particular with slightly charged areas of the lipids. And I'm not going to say if it's exactly 3.5, but between 4 and 5 k-cals, we can explain and understand why it's surprisingly cheap to insert charges in real membranes, although it would be quite expensive to insert them in pure lipid bilers.