And also, the channel's architecture is such that there is a cavity of water at the center. So when you look at the channel, what you realize is right at the point where the ion would be halfway across the membrane, there is a cavity of water, and the helices pointed with their C-terminal, or negative ends, towards the center of the cavity. In looking at that, you realize what the design is doing.

Again, this comes down to an issue of energetics. If you just think about bringing an ion from the water through a membrane, the channel somehow has to provide a pathway where the ion is energetically stable. One way it does it is by putting those oxygens that I have talked about in the selectivity filter. That's one part very near the outside edge of the channel. Right in the middle of the membrane, it doesn't have a selectivity filter, and that's in fact a place where, if you didn't have a special design, the ion, which is as we talked about most stable when it's surrounded by waters, is far away from the waters. So that's energetically unstable. But what the channel does is, it actually has a cavity of water in the middle, so it brought in water that stabilizes the ion at that point where it ordinarily would be farthest away from the water. So halfway across the membrane, there is a cavity of water, and then there are helices with their negative charges pointed at the cavity, and since positive attracts negative, that makes sense, because what it has is, in a sense, elements pointing partial negative charges to stabilize the cation [positive ion] at the center.

The cell membrane ordinarily would be a big energetic barrier for ions crossing the membrane, but the channel's design is such that this barrier was brought down, and the ion can easily slip through it. These features of the cavity and the helices were something that we never really could have predicted without seeing it.

Part II

SA: Generally, purifying proteins and then crystallizing them is an extremely tedious process. How long did this take you for the channel, and what was the most frustrating part of the process?

RM: Crystallizing proteins can be long and tedious, especially for what are called "membrane proteins," and ion channels are membrane proteins. Membrane proteins refer to proteins that are suspended in the cell membrane. Many proteins are floating in the water inside the cell, and those ones are generally easier to work with. The problem with membrane proteins is, their ends are pointed into the water on either side of the membrane, but the whole center of the protein is in the oily substance of the cell membrane.

In order to crystallize a protein, you have to take it and you have to first purify it in large quantity, and then you have to concentrate it and put it under conditions where it will organize into a crystal. Now, with a membrane protein suspended in the membrane, that's impossible unless you take it out of the membrane. I shouldn't say impossible; there is a technique where people make two-dimensional crystals, and they study those with the electron microscope. But the way we were approaching this problem is to make three-dimensional crystals, because if we could do that, we could solve the structure in a very straightforward way, if we could obtain good crystals.

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