 Hello, welcome back to today's discussion of metals in biology. In the last class, we were discussing calcium modulating protein right, CAM right calcium binding protein that means, calmodulin. This calmodulin the calcium modulated protein has 149 amino acid monomer and this is expressed in almost every eukaryotic cells and up to 1 percent of total protein mass binds 4 calcium ions that is quite a lot. So, protein mass we have 1 percent of it up to 1 percent of it is bound with calcium and this calcium binding gives rise to the dramatic conformational changes. These conformational changes allows the transmission of calcium signal. So, the moment calcium is bound with the protein that orientation change of protein structure will gives rise to the signal for another things to happen inside the cell or outside the cell. So, that is that is what we are we are going to going to discuss today. So, this is the closed structure of apochem that means, minus calcium when it binds with calcium as we have seen it gets really beautiful organized structure as you can see over here right. So, completely disorganized structure apochem becomes organized upon binding with calcium. If you zoom at this region carefully, we find that there is indeed quite exciting way of binding of calcium we have seen that also. So, this is the alpha helix, this is the loop and this is helix AF, so helix E loop and helix F. So, helix E helix F and the loop as if in on the right hand this calcium is bound as it is shown over here. So, this is called EF hand domain protein, this EF hand domain identified in the crystal structure first time for parabolamine and this helix loop this helix loop helix structure is quite exciting for a number of reason and that this calcium binding turn loop of this gives rise to the turn loop of nearly 9 residue and this always often occurs or always almost always occur in pairs right. And if you zoom further we can see that we have a calcium site that is where we were looking at more carefully and it is 7 coordinated as you may be able to see primary coordination sphere of calcium is having 7 coordination aspartate, men chain aspartate, aspartate, glutamate and water molecule is bound over there in this in this beautiful calcium binding mode of this of this calbodulin structure. Now, the there are many factors that we discussed that influence calcium to coordination to EF hand domain one of the thing is cooperativity one once one calcium binds another calcium binding occurs very quickly because it can orient the other structure or other site rather easily type of cooperativity what we have seen in hemoglobin cases it is similar to that where one of the binding all of the metal binding leads to the further increase in the binding or the binding constant becomes very high and the binding affinity goes up. So, another important factor that influence this calcium binding is the cellular magnesium ok. Now, this sort of binding is quite exciting for a number of reason because this calcium binding has physical consequence once this calcium is binding with the protein there is a signal that goes out right and these structural changes are also quite interesting and therefore, calcium overall is acting as a second messenger it is used the calcium is used to transmit a signal in a cell. So, this is showing saying that let us say calcium concentration has gone up once it is binding with the with the protein though. So, this signifies a number of things it may activate cellular components for example, enzyme some other enzyme can be activated and other cellular action may be triggered during this cascading signal process. Of course, most EF hand domain proteins are structured in the apostate binding of calcium from the apostate to binding of calcium as we were saying. So, apostate to the binding of calcium results in a change of configuration as you can clearly see over here change of conformation is happening these are huge changes of confirmation and therefore, this message or the change of conformation allows the calcium binding protein to transmit the message of an increased calcium concentration or to transmit this signal cam will then this cam will then binds to various target protein after calcium coordination. So, essentially in the nutshell what is happening is there is a protein in the apostate it is it is not too much doing anything at the moment the moment calcium concentration goes up or calcium binds with it then it gets a particular organized structure this calc cam or calcium bound cam now the hollow cam will be able to target different proteins and it will be able to bind with them these overall binding will gives rise to the many consequence inside the cell. For example, of course, it can activate a number of physiological function we will see briefly some of its activity for instance before getting into that let me say that this flexible alpha helix region this allows cam this overall as you can see 2 calcium over here another 2 calcium over there this flexible alpha helix allows hollow cam to clamp down on target and usually these targets are nearly 20 residue cationic and amphiphiotic regions of protein and these short of these short of alpha helix accommodate different other protein as you can see over here it is this protein side chain it clamps down and these clamping will trigger a number of physiological consequences. For example, it can activate camkinage too let us see that. So, this is there is inactive form of the enzyme this is activation of camkinage to pathway by calcium. So, this is the calmodulin as you can see 2 of the calcium here 2 of the calcium here can bind as is shown by this yellow dots and upon binding to this calmodulin this calcium loaded calmodulin now is ready the organized structure upon calcium binding is ready to clamp down on these inactive form of the camkinage. So, this is how it clamp down as you can see it is clamping down on these inactive form of the camkinage 2 resulted in activation of this enzyme. Once it is activated this auto phosphorylation takes place. So, ATP is converted to ADP this auto phosphorylation leads to the fully activated form of camkinage. So, without this calcium binding or calcium bound calmodulin this was inactive form. Once calcium is bound it becomes active and therefore, this is now very activated and this is converting ATP to ADP it is now in fully bound state. So, calcium 2 signal recognized and transmitted by cam and cam binding changes the conformation of camkinage and therefore, auto phosphorylation is occurring. Well and as you can see subsequently calcium can go out calmodulin can be released and again this cycle can go on of course, there is sometime the memory trace of friar calcium pulse also can lead to this overall camkinage 2 is present in the nervous system and concentrated at synapse and these are involved in learning and memory. So, you can see that calcium binding to calmodulin can have also effect in learning and memory of a given person right. So, the proper concentration of calcium or maintaining proper concentration of calcium is absolutely critical as you see without those calcium calmodulin will not be able to bind with camkinage 2 and therefore, activation of camkinage 2 will not happen. Upon calcium binding or suitable concentration of calcium binds with calmodulin that can clamp down further on the inactive form of the camkinage 2. This overall activation will lead to the phosphorylation or auto phosphorylation process where this camkinage is fully active such an active intermediate or active form of the enzyme will have direct impact in learning and memory and therefore, overall calcium can influence the learning process and the memory process again this is a camkinage 2 is a very very important enzyme which is found in nervous system and it is concentrated at the synapses. So, so far this sort of calcium binding can also be incorporated in a number of beautiful experiments. For instance, so these blue fluorescence protein and the green fluorescence protein they are separated from each other and attached with calmodulin ok. So, these two units are separated from each other and attached with calmodulin this is a research study which is showing clearly that these calmodulin attached these proteins can be brought together while calcium is binding with it. So, this calmodulin is modified of course, and two different proteins are attached with it. This calcium once it binds it folds as you have seen how it folds how it gives rise to the various organized structure overall that calcium binding and organization of the calmodulin gives rise to a situation where this calmodulin now organized and bringing these two fragment of the blue fluorescent protein and green fluorescent protein together resulting in these fluorescence resonance and energy transfer threat. This is quite phenomenal what essentially we are trying to say is the two proteins are separated or apart from each other they are they are linked by this calmodulin protein without calcium they are separated, but when calcium binding occurs this organization or reorganization of this calmodulin leads to a situation where these two protein which were far from each other they comes very close to each other and then there is fluorescence resonance energy transfer occur which can be monitored spectroscopically. And this sort of behavior I think it is quite phenomenal this sort of study to be able to do and demonstrate that how calcium binding can change the geometry and the overall disposition of the different fragment of the protein or different protein is quite phenomenal ok. Let us get into a next topic we will briefly discuss zinc finger domain right. So, that that is going to be the next topic that is going to be zinc finger domains well zinc finger domains in the zinc finger is required for or zinc is required for specific DNA binding ok. Now, there are there are protein that interacts with DNA in a extended manner, but during this interaction of protein with DNA what is essentially found if zinc is there that can give rise to the organized structure of protein and therefore, protein will be able to interact with DNA. Now, there is a specific motif at which this zinc finger domains are binding. So, tandem sequence binds zinc 2 with cis 2 and and his 2 motif. So, if you if you see in a protein backbone you know at f at from the N terminal 3 fourth of it exhibits 9 tandem repeats wherein 2 cysteine and 2 histidine are present. If you see this motif that is present this 2 cysteine 2 histidine motif which is repeated at the N terminal at the 3 fourth of it which is a 9 tandem repeats and this is overall can bind zinc effectively 2 cysteine and 2 histidine. Each tandem sequence binds zinc with 2 cysteine 2 and histidine 2 motif. The motif is of course, found in many other protein and zinc 2 binding with these 2 cysteine 2 histidine you know causes folding of the structure and that can bind then to DNA. So, essentially what we are trying to say is there is a protein which cannot interact with DNA effectively, but if the protein has a particular sequence as we have seen over here this sequence will be able to bind with zinc effectively to give rise to the ordered structure. Some sort of ordering as we have seen in the in the calcium binding. So, calcium binding gives rise to an ordered structure from a disorder protein backbone right. This ordering or the structure orientation gives rise to a situation where zinc will be bind in a completely organized fashion. This organized binding or organization of the protein upon zinc binding results in interaction of that protein with DNA right. So, let us look at that. So, this is how it looks like as you can see these are cysteine cysteine cysteine loop. This is bound with zinc, histidine histidine histidine loop overall it is also bound with zinc. This is a tetrahedral geometry we will come back to that and this without zinc this is completely disoriented structure right. This is the helical process this is cis cis loop and this is the linker. So, this compound formation has been characterized by various spectroscopic techniques. For example, this protein is taken and zinc is added and then the spectroscopic data are collected. So, in this zinc finger domain which is showing the zinc binding. The XF study EXAAFs XF study zinc 2 for zinc 2 plus K edge study shows that this zinc sulfur distance is 2.3 angstrom of course, crystal structure is not really initially known. This zinc sulfur distance or zinc cysteine distance is 2.3 angstrom this zinc nitrogen zinc histidine distance is 2 angstrom 2 angstrom and this is having a tetrahedral geometry. Of course, zinc 2 plus is detain system not many spectroscopic studies can be done when zinc is replaced by cobalt 2 plus which is having 3D7 electronic configuration when zinc is replaced by cobalt 2 plus it is also found that it is showing the tetrahedral geometry with cobalt 2 plus and it is coordinated with 2 cysteine and 2 histidine as shown in here. Of course, one can do also 2D NMR of the double standard beta sheet and alpha helix and this is also corroborating with this fact that this 2 cysteine and histidine is bound. Well, let us look at the crystal structure which obtains subsequently this crystal structure clearly shows that 2 cysteine and 2 histidine is bound with zinc in the zinc finger domain protein. That gives rise to a very beautiful understanding that the zinc finger proteins are going to bind in an order fashion this 2 cysteine and 2 histidine motif once it is found it will bind with it. There are number of experimental studies has also been done which clearly suggest that no formation of alpha helix in this alpha helix formation occurs in the absence of zinc 2 plus. So, if the zinc 2 plus is not there so, this alpha helix formation will not be happening and therefore, it is the overall protein cannot interact with DNA. Quite interestingly this cysteine residue coordinates with zinc first. So, the cysteine coordination with zinc occurs first and then the histidine coordination occurs. Of course, the alpha helix formation occurs prior to the zinc histidine bond formation essentially telling that this 2 cysteine binding over here occurs with zinc first and then this alpha helix formation happen and then histidine comes close to the zinc and then it binds. This is quite enormous I think that is that is sort of understanding how the ligation is happening with respect to the metal center is quite amazing. What we are trying to say is there is a protein backbone there is a 2 cysteine and 2 histidine motif the way we have discussed earlier this sequence is conserved. Now, once these 2 cysteine seeds zinc it binds with it, but of course, the alpha helix loop it has not been formed before that. After this cysteine binding to zinc then alpha helix structure forms that we are seeing over here. Now, upon alpha helix structure formation these histidine comes close to the zinc and then they bind. So, this binding first then alpha helix formation and then this histidine is coming close to it ok. Overall, the conclusion from many different experimentation and the molecular dynamic studies shows that the zinc coordination is required for folding of zinc finger to peptides right. Zinc coordination is absolutely required for perfect folding that would lead to the binding of these proteins with DNA. The peptide will be unfolded or adopt a completely different fold in the absence of zinc right ok. So, another question I think would like to answer then why it is so specific for zinc and why it is having highest affinity for zinc this zinc finger domain why not something like cobalt 2 plus which can have a you know very very good tetrahedral I mean you know dissociants once it is bound it could have a very very good orientation with respect to with respect to these 2 cysteine 2 histidine. Why it is show that zinc finger domain binds zincs specifically. So, the queries we are trying to answer is zinc finger domains, zinc finger domains display the highest affinity for zinc and why is that right. Well of course, different metal ions such as zinc, cobalt, nickel, iron is in plus 2 or plus 3 oxidation state can bind with with these 2 cysteine and 2 histidine motif. The dissociative value is quite high in fact, for cobalt, but if you look at the LFSE ligand field stabilization energy we will be perhaps be able to answer it is. So, consider ligand a ligand field stabilization energy for cobalt 2 plus and let us say zinc 2 plus coordination right. So, if you consider that you will immediately realize that cobalt 2 plus let us say cobalt hexa aqua complex. So, of course, no a metal ions are free it has to be depro it has to be it has to undergo dehydration to interact with the protein backbone. If you look at this is in octahedral geometry you will be able to see that it splits the d orbital and this is a d 7 geometry so, 5 here, 6 here, 7 here. So, overall if you calculate the ligand field stabilization energy it comes down to minus 4 by 5 delta 0 of course, plus 2 p pairing energy for both of them right. So, if you calculate it properly then this is of course, going to be plus 3 by 5 delta 0 this is going to be minus 2 by 5 delta 0 overall it is minus 5 4 by 5 delta 0. If you are calculating it for the tetrahedral cobalt species di cobalt cobalt dysistin histidine species then let us say let us draw the tetrahedral geometry you can have this overall 7 electrons are oriented in this fashion overall you can have minus 6 by 5 you can do the calculation yourself delta t and plus 2 p 2 p is small, but this delta t value is very small compared to delta 0. Although this is minus 6 by 5 delta t and this is minus 4 by 5 delta 0 there will be for cobalt there is a loss of 4.5 kcal per mole ligand field stabilization energy while going from octahedral to tetrahedral right. From octahedral to tetrahedral there is a loss of there is a loss of stabilization energy for zinc 2 plus there is actually there is no loss and that is because this is zinc 2 plus this is detent system from octahedral to tetrahedral there is no loss of loss of stabilization energy therefore, this can happen quite easily for zinc. So, what we have seen so far is zinc shows tremendous affinity to bind these two cysteine and two histidine and that is due to the fact that zinc is detain in nature. Some other metal centers such as cobalt originally these are hexa aqua cobalt complex 2 plus let us say they have high ligand field stabilization energy and they have tendency to stay in the octahedral state rather than in the tetrahedral state that is required for the zinc finger domain protein 2 histidine 2 cysteine to bind right. That is why this is very specific for zinc and zinc is quite happy over there and we have seen all over this, but quite interestingly we should also note that these sort of zinc binding calcium binding are giving rise to a situation where the protein is getting structured and can interact with a target protein or DNA or even give some sort of signal for us right. So, let us look at very quickly the metal mediated protein misfolding and diseases right. Of course, as you can as you know these sort of things are quite exciting and quite difficult to deal with because the understanding of these diseases are not much known right and these are very difficult disease if any anyone have seen any Alzheimer's disease patients and Parkinson's disease patients you understand that life is very difficult. And therefore, worldwide there is a lot of studies that is that is going on to understand how to provide solution for treatment and how to really better understand these diseases. What human diseases actually leads lot of human diseases are due to these folding or rather misfolding of the monomers protein. As you have seen both the calcium and the zinc can bind with the protein backbone in an organized fashion, but these sort of binding also mean that if they are not happening in an organized fashion the resultant can be the resulting picture can be different disease such as Alzheimer's disease as well as Parkinson's disease. So, as you can see over here this is a folded monomers and this is a unfolded monomers right. These are in always in equilibrium and these are misfolded monomers. This misfolded monomer can then go to the misfolded dimer then that can form fibrils, amyloids and insoluble deposits. These processes can be catalyzed by different metal ions right. As you have seen very recently that these metal ions can bind with these protein in different orientation there is protein backbone and protein side chain which can bind with this metal center and gives rise to the misfolding ok. Of course, they can also gives rise to the desired folding, but a lot of cases where misfolding is happening can gives rise to the many disease states including Alzheimer's and Parkinson's these are really difficult disease to cure at this point. Better understanding how to prevent these diseases definitely has to do how better we understand and control the metal binding and the detail understanding of the mechanism of this misfolding is going to be quite crucial in curing these disease. So, the role of metal ions in these processes of misfolding is oftentimes suggested and then of course, these remain an active area of study not much breakthrough chauffeur has been done. Hopefully in the years to come we as a scientific group will be able to solve some of these problem for the future generations. With this we will see you soon keep studying see you next.