 Friend of order might have complained when I show on the previous slide that I said that membrane proteins might insert either N to C, which is the obvious way, or with the C-terminus there. Now that's just for a single helix, but what if I have a large membrane protein? Let's draw the membrane again. Let's say that I had four helices. Then I can start on the, let's say that this is the inside, I can start on the inside, go out and in again, and then out and in again. Again, saying drawing N and C is a great way to indicate where the starter end is. Or maybe that protein should have the N-terminus there, and then go in, out, in again, and out one final time. It's going to be a pretty big difference here, right? Because if this is supposed to transport something in or out of the protein, you can't just invert it and expect it to have the same role. That's going to be problematic. So roughly at the same time where Don Engelman showed some of those simple results for helix packing, Gunnar von Heinen, here in Stockholm, that has to be a colleague of mine at Stockholm University, he came up with a very simple idea just by looking at protein sequences. So it turns out that while membrane proteins as a whole are mostly hydrophobic, remember how I said that the hydrophobic part was just inside the membrane, the helices? In these loops, you frequently have charges. And what Gunnar noticed is that there appears to be an offset here, so that you don't have random charges, but you tend to have positive charges on one side and negative charges on the other side of the loops. And in fact, what Gunnar showed that you overall, we tend to have positive charges on the inside. So if this on the inside of the cell, you would have more positive charges. You can have some negative charges, but you would have more positive charges there, and then maybe more negative charges on the outside. The way Gunnar was able to prove this. So first, the results we could see from bioinformatics that there was a pattern like this. What turns an interesting anecdote and hypothesis into an amazing biological result is that he then went into the lab, took this protein and swapped some charges to put the charged amino acids on another place in the sequence. And what he could then show is that in that case, the entire protein changed location so that you would have the previous inside on the outside and the previous outside on the inside. Why does that happen? Well, it turns out that most cells, virtually all cells in fact, have a so-called membrane potential that if I have a cell here, in general, it's roughly minus 100 millivolts charged on the inside. The electrostatic potential on the inside of cells is lower than it is on the outside. And that of course means that it's going to be physically advantageous to put positive charges to face the negative potential and the negative charges on the outside. Where does this potential come from? Well, that has to come from ions, right? Ions are really the only charged particles. So this means that we must have an excess of negatively charged ions on the outside, sorry, an excess of negatively charged ions on the outside or an excess of positively charged ions on the outside, meaning fewer positively charged on the inside. And it turns out to be the latter. Cells on average, we've pumped out positive ions so that we have a deficit of positively charged ions on the inside. And that brings us to something interesting. How on earth can we pump ions through a membrane? Ions are charged, right? We do not expect to be able to have something charged in the membrane. That is related to the causes of membrane transport that we will look at next.