 So how do we study this type of processes? Well in general it's hard but for the specific case of proteins that are folding there are a couple of very simple experiments many of them that we've already touched upon. In general I want to see can I find a simple measure that tells me how folded or unfolded is a protein. In this particular case it's a cd spectroscopy experiment I'll show you that on the next slide and then we're seeing here on the one side in this graph the protein is completely native and then I'm adding guinidinium hydrochloride we're moving to the right there and that's going to unfold the protein. If you look at cd spectroscopy in particular the one of the persons who initiated this was Christian Anfinsen that you know this is a picture when he's slightly older when you saw him last time. Remember that Christian worked on this ribonuclease that caused him to understand that proteins really must, proteins are folded based solely on the amino acid sequence and therefore the native state must be the thermodynamic free energy minimum. The way he started this was part of by using cd spectroscopy so cd stands for circular dichroism and in a way it's a somewhat fancy way of describing working with polarized light. Now for technical reasons we don't work with horizontal and vertical polarized light but we tend to work with circular polarized light meaning that when they're the vertical and horizontal components are offset from each other by a quarter of a face. Depending on the order they occur that means that the light will either be right polarized or left polarized. If we then submit beams that occur that contain both right and left polarized lights through a sample what I can then study is that the difference in these beams so for instance how much are we observing the right polarized light versus the left polarized light and that's going to lead to instead of the net amount being plus minus zero that would correspond to say having slightly more right polarized or slightly more left polarized just as if I'm turning an angle and that's exactly how you measure it. So in a way we just need some circular polarizing filter that's a couple of dollars and then I need a detector and then I need a computer today but this is an exceptionally simple device. The reason why this works is that our proteins amino acids in particular are chiral you remember that you can't take the mirror image you can't take the mirror image of it but I can't rotate it back to the original. That means that the chiral centers here will have a pattern in how they absorb right versus left circular polarized light and that in turn means that the entire secondary structure elements will have a particular pattern of how they absorb light. That in particular means that if I compare say an alpha helix to a beta sheet to a turn they're going to have different spectra in this particular case we're measuring this so called ellipticity that is how much I'm turning the light as a function of the frequency of the light that I have on the x-axis. Red curve is alpha helix green curve is beta sheet and the blue one here is a coil so very clear pattern and by then by looking at different wavelengths here I can easily fit how much helix how much sheet and how much coil do I have in my sample. Now if I do that type of experiment but then say try this at different temperatures at each temperature I might have a mix but then I can see what's happening to the protein structure overall in this particular case I might not be interested to know that I have 15% helix but have I destroyed the secondary structure or not and that you see here one experiment but at three different temperatures at each temperature here I likely could fit this to the three curves below and say how much helix sheet or coil we have. This is a fantastic experiment the only single drawback is that I only get the total amount of helix I can't say that I have a helix from residue 17 to 25 if you absolutely need that the first thing you should do is probably buy informatics and the second one would be NMR spectroscopy. What on earth do I want to use this for hadn't I introduced this already well I had but what we haven't covered I'm not sure whether you're aware of that I talked about these phase transitions I haven't proved that protein folding is a phase transition maybe you took it for granted maybe you thought that that was a deviation about physics but the folding of these small domains that we've talked about it is a phase transition and it turns out that we can prove that.