Cryoprotectant Molecules - Nature's Anti-freeze

Chris -  So how are you approaching this problem?

Lorna -  Well Chris, I'm a physicist using experimental techniques to try and understand how cryoprotectant molecules work.  Cryprotectant molecules are things like glucose and glycerol, sugars and sugar alcohols, and they're used around the world by people in research labs when people want to store proteins or cells in fridges or freezers at low temperatures for long periods of time.  Having the sugar or sugar alcohol there allows you to do that - it allows you to bring those proteins or cells to very low temperatures without them being damaged.  So in my research lab, we're trying to figure out how it's possible to do that, and the particular area that we're focused on is proteins. Now proteins have this very unique three-dimensional structure, and that structure is really important for their function.  When you heat a protein up or apply a force to it, you unravel it, just like you would unravel a piece of string, so you reduce that three-dimensional structure.  What we want to try to figure out is what happens when you add these interesting cryoprotectant molecules to the surroundings of the protein - what do they do to stop the protein unravelling?

Chris -  So at the moment, we're using these chemicals because we know they work - but scientifically speaking, we don't really understand very much about how they're working?

Lorna -  That's absolutely right. They work, so why do I need to do these experiments?  One of the things that's motivating me is some research that's going on here at Leeds where they're using these cryoprotectant molecules to preserve ovarian cell tissue. This is incredibly important for fertility treatment.  Now the problem with this is, if you use a molecule like glucose or glycerol to preserve these cells, when you freeze them and then rethaw them at a later date you only recover a certain percentage of the cells.  Ideally, we would want to recover as much as possible so that we can use them for later treatment, so we want to go right back to the beginning.  We want to understand how these molecules are working, we want to understand how different cryoprotectant molecules work, and then we want to figure out which are the right cryoprotectants to use for particular proteins, cells and tissues.  So we really want to get down to the details of how this mechanism works.

Chris -  What do we know so far about how it's working? Because we've got lots of natural examples to look at: there are plants that grow in very dry countries that can survive enormous amounts of desiccation, and they stabilise themselves and come back to life as soon as they're wet again.  There are other things that can survive temperature extremes in the opposite direction.  Are there any chemical processes that unite the two that can give us a clue as to how these organisms are able to resist these extremes?

Lorna -  Well one particular school of thought is that the cryoprotectants do something interesting to the water in the environment.  So, for example, glycerol can do something to stop the water freezing - when water freezes, it forms this extended hydrogen bonded network, and this can be incredibly damaging for biological cells because it can rupture the membrane of the cell.  That's one line of research that we're following -  we're trying to look at the details of the water network when it has cryoprotectant molecules in its vicinity, and it's really not that simple, as it destroys the hydrogen bonded network.  It's much more detailed than that, so water is still able to form a network, but glycerol for example is very effective at getting in between that hydrogen bonded network.  So certainly, it is diminished and in this way, it could stop the water freezing at its normal temperature. If you look at the freezing temperature of water and glycerol, water freezes around zero degrees.  Glycerol freezes at about 20 degrees, but if you put them together in a very particular combination, you can get that mixture to freeze at below zero degrees, right down at minus 47 degrees.  So there's something really interesting going on when you mix these two hydrogen bonded liquids together.

Chris -  It sounds elegantly simple, but I'm sure it's a totally different matter to try to work out the movements of particles which are literally just a couple of atoms glued together.  So how are you following these networks of particles, the water molecules, and the glycerol, and seeing how they interact together?

Lorna -  We're using two experimental techniques.  The first one is neutron diffraction, and we're doing those experiments at the ISIS facility at the Rutherford Appleton Laboratories near Oxford.  Neutron diffraction allows you to look at atomistic level detail at liquids and so, it allows us to ask these questions about the hydrogen bonded network.  Of course, we're also interested in proteins, and we're using another experimental technique to look at protein stability.  We're using an atomic force microscope which you may have heard of before in the show.  This instrument was created back in the 1980s, and it actually earned its creators the Nobel Prize in physics.  We've built a modified form of the atomic force microscope here in Leeds, and what that allows us to do is pick up a single protein and apply a mechanical force to that protein. That force is enough to unravel its folded 3D structure.  It's a very specialist piece of equipment because the forces that we need to apply are actually piconewtons and we're working with molecules that are on a nanometre length scale, but we do this every day in the lab.  So we're applying these tiny forces to proteins in the presence of cryoprotectant molecules, and this allows us to look at the details of the kinetics and the dynamics of protein unfolding and folding in these interesting cryoprotecting environments.

Chris -  And so, putting it all together for us Lorna, what do you actually think is going on when we mix one of these cryoprotecting chemicals with say, a cell or a set of proteins to protect that protein down at very low temperature?  

Lorna -  I think that the solution is incredibly important, so the hydrogen bonded network that the water is forming plays a massive impact on the stability of the protein.  I think that where the cryoprotectant molecule is interacting with the cell or the protein itself can give it extra stability as well, and the concentration of that solution is key to the temperature at which you can reduce the system without it becoming denatured.