 So, we have been talking about some aspects of protein dynamics, we said in proteins the dynamics can occur at various time scales which I am showing this slide once more, various time scales are there for protein motions, they range from picoseconds to the minutes and hours and of course, this protein dynamics itself is a huge subject, one can talk about it for several hours, but here we are going to take a few of those topics and discuss in some detail. And I already talked about the particular aspect of millisecond to microsecond time scale motions, these are in conformational exchanges, two side chemical exchange, this we talked about last time and also how depending upon the exchange rate and the chemical shifts of the individual sites, the lines will change in character, the line widths will change how they merge depending upon the exchange rate we service the chemical shift difference between the two. We are going to look at one more aspect of this time scale motion which is on the minutes to hours time scales and this particular thing is also related to protein stability and folding unfolding. So this has implications for proteins stability, folding, unfolding. And what are you talking about? This is the motions on minutes to hours time scales and sometimes even seconds to you can take minutes time scales and this is what is called as NH deuterium hydrogen exchange. This is HD exchange and what is exchanging? This is the NH protons. So NH protons, amide protons they exchange with D2O, when you put D2O, this forms ND plus HDO. This is what we are talking about. So you have a protein, let us say various kinds of a protein here and there are various protons sticking around here, proton here, proton here, proton here. And this is a folded protein, some hydrogens are on the surface, some are in the interior and then when you put D2O in it, some of those which are on the surface they will exchange and the ones which are in the interior will take time. So there is a graded exchange NH protons and this is intimately connected with the local folding unfolding, this is intimately connected with local slash global unfolding events. Therefore this has implications for protein stability. So this became an important technique hydrogen exchange, this is also called as HX experiments. These are also called as HX experiments. So the idea is that you monitor the exchange of the amide protons with D2O. And that has implications or it will indicate the relative stability of different portions of the protein. I will show you this here. Suppose you have a HD exchange, you have here a protein which is like this, it has a helix here and it has various sheets here, beta sheets and there are a lot of protons sticking around. Now here the red ones are the deuterium and this ones are the protons. Now if you put D2O to a solution in water, you have the protein dissolved in water. So therefore initially all the protons are in the H state, you can lipolyse it. So when you lipolyse it, make it dry powder. Then it is all H and H. Now it is dissolved in D2O, you add D2O to it, add D2O to it. What will happen? The ones which are on the surface of the protein, they will exchange immediately. So you start getting here these red balls, the red balls are the deuteriums. And this depends upon time. How much time you give for this? You keep D2O inside, you monitor as a function of time, monitor as a function of time. The ones which are on the surface will immediately go away, from which are in the interior they are not ready to exchange immediately. It requires a certain process for them to get become accessible, then you go on with the time. So more will get deuterated, for this will actually reflect in your NMR spectra. How do you monitor? Where do you monitor? You monitor in the HSQC spectrum, proton N15 HSQC spectrum. So this is the spectrum or the HMQC spectrum, you can use either the HSQC spectrum or the HMQC spectrum, it does not matter. So this is the initial state of the protein where it is all protonated. This is not deuterium. Now you see as a function of time, you add D2O to it, as a function of time you can see here, this is 9 minutes, this is 63 minutes and this is 133 minutes. You can see how this number of peaks starts going down. Why do they start going down? Because we are looking at proton N15 HSQC. What we are looking at is spectrum is proton N15 HSQC spectrum. So wherever there is a proton, it will show a signal. If there is a deuteron, it will not produce a signal. The ones which are exchanged out, the ones who are exchanged out with the deuterium, all these red ones will not show your peak. So as a function of time, if you look at the spectra, you see the peaks will start disappearing more and more, showing that these ones are in the interior, some of these are still there, everything has not gone. Even after this many minutes, this is after 133 minutes, this is almost more than 2 hours, all of them have not gone. So some and slowly, slowly as we increase the time, more and more are disappearing. From here to here, some disappeared, here to here, more disappeared, here to here, even more disappeared, you keep going. In certain proteins, the proteins are so stable, it will take years for them to completely exchange. What is required here? What is required is that there has to be some local unfolding events, because the protein is in a folded state. If the protein is in a folded state, the NH proton, this is a folded state, it is not accessible and this has to become unfolded, unfold. Then after that it can exchange with V2O, then it can end there. So there is a process here, there is a folding unfolding process. So depending upon which one is how the local folding unfolding events are happening, you can have different kinds of exchange rates. And therefore, you will have, we will come back into this little bit more detail. And this is the process that happens, that is why you see some peaks are remaining and more and more disappear as you give more time. So local folding unfolding events continuously keep happening, although the protein is not totally unfolded. It is still a folded protein, but there are local dynamic changes that are happening. The dynamic changes that happen inside make the proton accessible to the D2O which can actually penetrate this protein structure and then it will start a exchange process. So then what you do is, and as you also keep looking at this, even here, see you see these intensities of the particular peaks, even those peaks which are present here, the intensities will keep decreasing, do a function of time, we do it for many times. We have shown only the four time points here, but you do it for many time points, you can monitor the intensity changes of all of these peaks, how they eventually disappear. So if you plot these intensities, this is what is shown here. So as a function of time, you plot the intensities of the peaks. This is shown for a few residues here. This is one, there are three residues, we have shown a particular protein, it does not matter which kind of what protein it is. But three particular proteins are shown, you see this one slowly exchanges, these ones are very rapid. This is the most rapid, after that this one, after that this one. So there is a clear variation in the exchange rates of these ones. This exchange is very rapidly, very likely this is on the surface, the one which is here and this one is probably slightly in the interior, it takes more time. Initially all of them will start at the same point. And then this one is very slow, so therefore this is in interior. Now what you do? You fit these two functions of this type, exponential functions, fit this to an exponential function either this or this, you can fit single exponential function or a biexponential. So this will indicate the number of processes that are happening in the process of exchange of the protons. So this is the single exchange here, so it gives you, there is one exchange rate indicating one process, here there are two exchange rates indicating two processes, this may be local followed by global and things like that. So all of such things can happen and that will result in different kinds of fitting parameters. And these ones are important, obviously these exchange rates have information about the structure of the protein. You see here, this is for a particular protein here. What is plotted here is the log of the exchange rate versus the residue for a particular protein. Now these ones are very fast exchanging, these are very fast exchanging, exchange rates are very high. Then you have these slow ones here and you see a variation in the exchange rates. And notice the ones which are in the n-terminal or in the loop area, these are the secondary structure elements in the protein. You have a helix one here, there is a turn here, there is a helix, there is a helix and there is a helix. These are secondary structures and they are connected by certain loops and turns. The way there is a turn, actually the possibility of it is getting exposed to the solvent is more therefore they will exchange very rapidly. Therefore the exchange rate is high for those ones and where there is a regular secondary structure then you see that the exchange rates are relatively small. Therefore this is indications about the structure of the protein, the stability of the protein, different areas of the protein. Even in the different area, in the folded areas there is a variation in this exchange rates. So that will tell you how the protein can sequentially unfold and the stability of the protein can measure the equilibrium constants for the unfolding reactions in this place. That indication is already there. Now the same thing I am going to explain here to you and here is the process, this is the A and the H, this is probably A will be the amide proton here and this exchange can be catalyzed by certain kinds of catalysts and so you have the water, this is from the water which is coming the D2O and you have the OD minus, the A minus plus HOD and A minus plus D3O plus, this is at a particular pH, you have the hydronium ion here, the deutronium ion here, you get D2O plus AD, this is how the exchange process happens. And that is indicated here in a schematic manner with respect to the protein structure. You have a helix here and then you have a loop somewhat hanging around here and there is a proton here. Now this one unfolds, see this proton, now you see the chain unfolds here, this one unfolds, this portion remain the same, this unfolds, the proton is now becoming free. So there is an equilibrium here, the opening rate and the closing rate, there is an equilibrium here and then once it is in the open state then it will immediately exchange, this exchange rate is very high compared to this closing rate. Therefore once it is here, once it comes here, this exchange will happen in this manner. Therefore it is a relative importance of this rate versus this rate which determines how fast the system will exchange with the D2O. Therefore obviously it has to do with the equilibrium between these two here and that is indicated here. So if you have the unfolding like this, local unfolding, this is the unfolding rate, this is the folding rate and then you have this, once it is unfolded here, this exchange will happen immediately with the D2O, you get an unfolded protein. If you have an unfolded chain here, this is a partially unfolded here, so you have a certain segment, you can also have situations like this, it can go from here to here, from here to here and then here to here or it can go directly from here to here. So therefore you have the measured exchange rate is quantified in this manner. So there are two kinds of mechanisms which are indicated here is EX1 and EX2. EX1, this typically this closing rate is much much smaller than the exchange rate, that is this rate is much much smaller than the closing rate and which case that is called as the EX1 mechanism, once the protein comes here this will immediately exchange, so that is this situation as well. On the other hand there is the EX2 mechanism, this rate is much larger, this rate is much larger than this. Therefore the protein would like to remain in this state as long as possible. Therefore occasionally it will exchange because there is always a possibility, there is a certain chance, this is all kinetics, therefore one knows this, there always a certain probability and then the measured exchange rate will be given by this. So you have the K opening multiplied by K exchange here that is KCH and then the K close plus KCH plus K opening and this is given by K opening to KCH by KCL plus KCH. This rate if it is depending upon what you have, what relative magnitudes of this and this you have, so you have here different situations. For the EX1 mechanism where the K close is much much smaller than KCH what you can do is you can ignore this. Then it will become simply equal to K opening rate that is this one, so immediately goes here and then goes there. On the EX2 mechanism when the K close is much much larger than KCH then the K observed will be K open divided by, opening divided by K close multiplied by KCH. Notice here this is your equilibrium constant, K opening that is this, this rate divided by this rate there is the equilibrium constant. Therefore there is information about the equilibrium constant here for particular residue depending upon which residue you are monitoring you have the information about the equilibrium constant for that particular event. And therefore you can translate that into free energy and it gives you information about the stability of the protein. So therefore this is enormous possibility of determining the stability of the protein using the dynamics. So here we are talking about the dynamics, the kinetic exchange and that is a dynamic process happening at a millisecond or seconds to hours to sometimes even years time scales. Some of them remain in BPTI for example the protein stays for several couple of years without getting exchanged. There are some very stable proteins like that. Now this exchange is a catalytic process and depending upon the pH of the solution the exchange rate can be different. Now you can have catalysis process can be catalyzed both by the acid catalysis or by the base catalysis this can happen and in either case therefore you see there is a high exchange rate here and then you have a minimum somewhere here, somewhere around of course it can vary from protein to protein. This is not that it is all this is the schematic this is an indicator. You will have some below this is acid catalysed process that is the KCH we are talking about the CH when you have that this is acid catalysed and this here is base catalysed and now here you have the lowest exchange rate we are going to use this. This strategy one can use to get information about the protein folding unfolding events. Now when you want to do a protein folding study so either you can start with a folded protein and slowly go into the unfolded state or you can start when an unfolded protein go into the folded state. How do you do this? So you do this by kind of a denaturance using use of the denaturance. So you denaturance what is the effect of the denaturance? So one of the denaturant which is commonly used is urea and you can monitor the protein folding by measuring the fluorescence of the particular tryptophan or something inside your protein and as the protein unfolds the fluorescence will change. So now here you see this is the urea concentration you plot you monitor the folding unfolding process. The unfolded protein here your particular fluorescence intensity and the unfolded state you have the fluorescence intensity here and this is the transition zone typically you have the midpoint is somewhere here but sometimes of course this is a very schematic it can be for different protein different places this is at a particular pH this is for egg white and egg white lysosamide pH 3 but for different proteins it can be different the midpoint can be at 4 it can be at 3.5 and so on so forth at urea concentration in molar. The point is it has a kind of a sigmoidal behavior here in this state therefore typically if you are at lower urea concentration reasonably a wide range of urea concentration the protein can remain in the folded state we are going to make use of these to study the folding pathway of a particular protein. And from this EX2 mechanism which I indicated to you earlier that you can actually calculate the delta G the free energy of the folding unfolding event. So you have see in the folded state the free energy is very high this is in kilo joule per mole so you convert that into kilo calories of course you divided by 4.18. So therefore you will have here about 4 to 5 kilo calories per mole here and here it will be much less and this will be even much less here. So therefore the folded protein the folding to unfolding requires a larger free energy as a larger free energy here and then slowly it goes over into the intermediate free energy then it is a very low free energy for the state. So therefore this is typically when it is in this state the protein is able to exchange with the D2O much more rapidly. How do we use this? This we use for studying the folding mechanism pathway, protein folding pathway. What you do is you create an unfolded state, create an unfolded state by adding urea or something and then here the protein is completely deuterated. What you do? You prepare a buffer in D2O in urea and then of course you enlivenize the sample so then you get a powder and then you have protein which is deuterated but unfolded. Now what you do? Suppose I have urea as 8 molar, suppose I have 8 molar urea and then of course in this 8 molar now I have to initiate the folding process. This is an unfolded state, I want to go from here to the folded state. How do I initiate the folded state? Now I add in H2O, I add H2O to it dilute in H2O you add something like about 20 times dilution let us say. If you do 20 times dilution initially let us say you had 8 molar urea. Suppose you had 8 molar urea this is the unfolded state and you do let us say 20 times dilution in H2O but at low pH and what pH? This is at pH 3 somewhere around pH 2.5 to 3 you do at low pH. So then 20 times dilution meaning 20 fold dilution what will be the concentration here? So this will be like 0.4 molar urea. So 10 times will be 0.8 molar, 20 times will be 0.4 molar urea. Now you see at 0.4 molar urea the protein is still is folded it is not unfolded. So I mean it has not folded back it is in this condition only. Now what it can in principle fold. So this is what the initial folding event you start with an unfolded state now you initiate the folding by 20 times dilution with H2O that means you are providing a condition for the protein to fold but you are not providing a condition to exchange because you have put a low pH therefore the protein starts to fold for a certain period time you give a certain period of time it has folded to some extent but it is still D2O it is still deuterium because you have put the pH very low. So it is when the pH very low that is at around where that is the minimum exchange rate is minimum so it is still in the D2O. Now what you do is deuterium now you put a pulse label what is the pulse label here you put a pulse of water H2O. Then you put the pulse it means you put a increase the pH you increase the pH here at this point in time that means it is a kind of a pulse increase the pH you allow the system to start exchanging. So at low pH it is not exchanging you increase the pH at time tf for this until time tf you allow the protein to fold but it has not exchanged but at this point you increase the pH of the solution by adding a certain degree certain amount of hydroxide or things like that NaOH or something like that that means you have provided a condition for the protein to exchange from deuterium to proton. Therefore now you see whatever is exchangeable at this point it will exchange now this will become what is in the surface here suppose it has folded like this whatever are the deuterons here they will exchange to protons here they will exchange to protons. Now you want to monitor this so what you do? I want to monitor which protons have exchanged which deuterons have exchanged into protons therefore again I stop labeling that means I lower the pH once more I immediately lower the pH so that the exchange process is quenched. So no more exchange happens at this point so therefore what I have done I have given allowed the protein to fold for a period of time tf then increase the pH suddenly allow the exchange to happen for a certain time period and this also can be varied and then at this point at tp you stop the exchange process so suddenly lower the pH so during this between tf and tp the protons are exchanged the deuterons are exchanging into the protons stop labeling now you monitor this low pH and whatever has happened here you can read by two dimensional NMR in the HSQC spectrum you will start seeing whatever changes have happened here. So that is how you get the information about stepwise folding you can keep varying this tf keep varying this tf and tp. So if you give more time for this more protons will get exchanged less time less protons will get exchanged therefore you have to vary this so there are two things which one has to vary one is the extent of folding that is governed by this tf how much time you give and then how much at what time you increase and for how long you will keep the pH increased at a higher value so that the exchange happens and for how long you will keep it that depends upon how much how many protons will appear as NH protons and not deuterium therefore you can monitor this by 2D experiment in the HSQC spectrum because in the HSQC spectrum you will only see which are exchange you do not see the other ones ok. So that is how you monitor the proteins folding process the folding process you first denature the protein and initiate the folding process by diluting the denaturant and you allow it for a certain period of time and then you add you change the pH condition this of course has to be done very rapidly this has to be done very rapidly inside the solution itself you have to devise you have to design a certain hardware also for doing this because you have to give a pulse suddenly you have to give a pulse you add an NaOH so that the immediately the whole and the stirring mechanism also has to be introduced so that the exchange process starts and then after that you stop the exchange and monitor what how many protons how many deuterons are exchanged into you can keep varying you can be varying these times tf and tp ok. So this is how you this is hydrogen exchange hydrogen deuterium exchange can be used to study protein folding protein stability and and this time scales can be in seconds to hours time scales ok. So now we will use another strategy to monitor the folding of the proteins and this will be again start from the denatured state and we go over by monitoring systematic changes in the conditions of of the solution with regard to the urea concentration and that will be demonstrated I will see demonstrate with regard to the particular protein called sumo and what is shown here is the folding funnel and we will talk about that in the next class.