 Hi, welcome back to the fourth class of metals in biology. I hope you guys are studying all of you from the books available and some of the references that we have given during our lecture. One of the book that we are following is Principles of Bioinorganic Chemistry by Lippard and Berg any other bioinorganic chemistry book another exciting one would be the one by Professor Keim. So, please study from any book if possible try to study from these two books. In any case we were in the last class we are trying to discuss metal regulation and gene expression. Now, there are consequences as we are mentioning that the iron metal iron or metal iron concentration at very high level or at a very low level will have its implication in our health. So, for example, there is iron regulatory proteins which controls both ferritin which is a iron storage protein as well as transferrin which we were discussing in the last class translation. So, iron regulatory proteins IRPs in short they control both the ferritin and transferrin translation that is quite interesting right. Well by this way they can control the delivery and storage of the given metal iron such as iron as I was saying for iron regulatory protein. Regulation of toxic metal ions are essentially very important body ensures that no toxic metal ion even if it is internalized stage in the body for long. So, there will be a process by which toxic metal ion will be excreted or will get rid of those such as such toxic metal ion such as mercury will be thrown out of the body. So, that sort of regulation is also present. Well zinc finger protein overall controls the transcripts and process as I was mentioning IRPs controls the ferritin and transferrin translation we will see also in the subs in this class that calcium 2 plus which is a secondary messenger and found at the centriole of at the synapse the will also be important in controlling different metal ion concentration. Today we are going to first discuss the regulation of iron levels in cell. Regulation also has to do with the storage as I mentioned the storage protein for iron is ferritin the protein name is ferritin. It can store a huge number of iron centers while iron storage protein has 24 subunits and each of them are having 175 amino acid approximately. It has cubic symmetry apopheritin means minus the iron center of the ferritin where no iron center is bound apopheritin can house 1000 iron atoms in the central core. A ferroxidase center usually loads the iron into the apopheritin. So, there is a protein ferroxidase which is involved in loading the iron into the apopheritin so that ferritin can store the iron center. So, this is something like a relay mechanism. So, ferroxidase catches the iron and then it delivers to the ferritin. So, the transferrin the uptake protein that we have discussed separately ferroxidase is the transfer protein. Ferritin keeps and releases to ferroxidase and ferroxidase is loads it into the ferritin. Now, metalloregulation as we were talking in bacteria it occurs at the transcription level. So, DNA to mRNA formation at transcription level in bacteria these metalloregulations are occurring. In mammals the synthesis of apopheritin and of the transferrin receptors are regulated at the level of translation remind you not at the level of transcription. During mRNA to protein formation that is the translation step in mammals the synthesis of apopheritin and of the transferrin receptors are regulated. Now, as we know these iron ferritin subunits are going to have a quite complex structure all these exact structure although some crystal structure are known, but it is you know nailing down the 1000 iron loaded irons a ferritin center is not very easy. Of course, apopheritin structure are known which is which is having a 3-fold channel and also 4-fold channel overall there are many subunit as we have discussed and it can load the iron center in the ferritin. Now, let us look at briefly the ferroxidase centers which is known the crystal structure is known. Let us look at the apopheroxidase where you see over here this is the apopheroxidase structure these the site A and B this is taken from this book A and B can load or can house 2 different iron center in this site. This is the iron loaded form on the top as you can see the glutamate is bridging between these 2 iron center this is ferroxidase remind you ferroxidase loads iron center into the ferritin which is a storage protein. Now, this is capable of binding iron center this ferroxidase you see one histidine one glutamate another bridged glutamate water molecule another glutamate over there these are hydrogen bonded with tyrosine and even this water molecule as you may know will also be hydrogen bonded overall it is a huge network essentially it tells you that in ferroxidase 2 iron center can be incorporated and how their structure is going to look like this gives you a clear idea. Of course, ferroxidase is not ferritin ferritin is the storage protein ferroxidase is the one which is helping in transporting in a way of course, transporter is essentially the transferrin, but overall from transferrin ferroxidase is helping in loading the ferritin. Now, ferritin structure at a very simplified iron binding site is not really a good site what we have we are we can see for example, is the one where we have these you know synthesis attempt this is a science paper in 1993 which clearly shows that how perhaps multiple iron center can form a nice structure a mixed valent poly iron oxo cluster you know as this can be a model of ferritin core and this is somewhat perhaps the ferritin would look like of course, this is a model core model structure which shows that how perhaps ferritin might will look like in the in the in the ferritin ok. So, a lot of iron loaded sites are seen in this see these are mixed valent poly iron oxo clusters. Well in the metallo regulation then we have learned that in bacteria a single protein that is for iron uptake regulator in abbreviation called FAR FUR a single protein that is FUR FAR controls the transcriptions of genes involved in psi dero 4 biosynthesis ok. As we have mentioned that in bacteria the control material regulation happens at the transcription level and that is at that level the psi dero 4 which we are discussing in the last class controls its biosynthesis is controlled ok. Psi dero 4 biosynthesis is controlled therefore, iron uptake is also controlled at the transcription level. The protein that is involved in this process in bacteria is FUR for iron uptake regulator. FAR is a dimer with subunits something like 17 kilo Dalton at high iron levels the FAR protein has bound metal and interacts specifically with DNA refreshing the transcription of course. So, FUR protein the FAR protein has very high levels of iron. This FAR iron protein has metal bound in them and they will interact with the DNA specifically and therefore, these you know transcription can be controlled or replaced. So, overall what is happening is this FAR protein in FUR FAR protein in bacteria it is loaded with metal it prevents the DNA to RNA formation unless it is necessary. So, the high metal ion concentration of FAR will control the transcription or will repress the transcription. In bacteria that is how metallo regulation is happening and that is how the psi dero 4 biosynthesis is getting controlled. If psi dero 4 biosynthesis is getting controlled let us say then iron uptake will not be uncontrolled will also have control on the iron uptake. In mammals expression of ferritin and the transferring ferritin is the storage protein transferrin is the transferring protein which is which is involved in transporting iron as well as ferroxidase. So, expression of ferritin and the transferrin receptor is regulated at the translation level. So, overall as you can see there is lot of checks and balance which controls the metal ion transport when the metal ion has to be transported inside the cell that gets controlled by other protein either at the transcription level or at the translation level. In mammals it is at the translational level where it gets controlled at bacteria the biosynthesis of different psi dero 4 which is responsible for transporting a metal center let us say iron their control is happening at the transcription level. Now, we will discuss little more on these topic and we will come back soon. So, we were discussing the metallo regulation metal it is important to control to be controlled also metal plays a vital role in folding of biopolymers. Let us now try to see the metal folding of biopolymers the role of metal in folding the biopolymers. So, biopolymers such as protein as you know metal can bind with protein at different sites they are polypeptide proteins right. Now, this peptide has amino acids. So, the amino acid residue can bind with the metal ion and therefore, the structure of the protein can be modulated by a given metal ion. Let us say you have a very high coordination number containing metal ion. It will fold the metal protein differently compared to the low coordination one. Let us say you have a metal ion which can coordinate with the different side chain in an tetrahedral fashion or in an octahedral fashion or in a square planar fashion depending on that we can pretty much say that metal ions can fold biopolymers differently. Different metals can fold biopolymers differently depending on the metal ions its charge size as well as the coordination number the biopolymers can be folded differently. So, it is essentially what we see that if these metal ions are not the right one or excess of metal ions are present or less of metal ions are present the folding of biopolymers will change and therefore, the change of this folding pattern can also lead to the agglomeration or precipitation of the biopolymer and therefore, some of the disease such as you know Alzheimer's disease and Parkinson's disease can be directly be associated with the adventurous chemistry simple chemistry coordination chemistry that this metal ion can play. Let us look at the few principles for metal folding in case of the biopolymers. Well metal ions simply can act as template to organize the 3D structure of biomolecules that is what we were discussing now right. It can act as template for the 3D structure of biomolecules. Now depending on the necessity metal ion can lose water molecule when binding to proteins. Let us say you have a hexa aqua metal ions of course, no metal ions are completely free it has aqua molecule. If need be only 3 of them or 4 of the water molecule can be chopped off or can be dissociated from the metal center and the protein can come out. Selectively any given number of water molecule can be lost from the metal side and then the binding of the protein can therefore, be affected by these loss depending on the proteins we can have different orientation with respect to the metal ions. Metal ions can retain many water molecule can lose many water molecule and also can retain many water molecule when binding to nucleic acid and therefore, these are the processes which will also affect the hydrogen bonding right. Metal ions binding can also facilitate interaction between biopolymers. Let us say two biopolymers are there they are not interacting with each other at all a metal ion in between can act as a communicator or some other side reaction other you know unwanted things can also happen. So, two biopolymers multiple biopolymers can be interlinked through the metal center. It is a center of attraction for the biopolymers. Biopolymers feels attracted towards the metal ion because metal ion can bind with them right in a coordination atmosphere. Metal ions with large ionic radii utilize high coordination number for its function right. Metal ions with low size or low ionic radii will usually have low coordination number for its function. As you can see metal can interact with the polymer biopolymer. Therefore, it has also the ability to induce protein misfolding which may be relevant as we mentioned for different human diseases. So, overall we will also try to discuss let us say at least one thing for sure the calcium binding proteins and their EF hand domains in a moment. And also we will see if we would like to discuss the metal mediated protein misfolding and the different disease states. Overall this metal binding is crucial in case of conformational changes that can happen in the protein metal binding and this can lead to the biological function. Well we will be definitely discussing this in detail. Let me tell you that protein folding is quite important to understand. We must see how do peptides are folding and proteins are folding. Now imagine that we have a situation where only one bed is there and one person is sleeping. Then therefore, the person can orient himself or herself the way he or she wants this is a very relaxed situation. But on the same bed if 10 people has to sleep now they will feel like disturbed and therefore, the orientation of their sleeping mode will be completely different. So, in a crowded atmosphere versus a non-crowded atmosphere the protein will fold differently. But more importantly when we have a metal ions incorporated into the crowded atmosphere of the protein which is usually the case in a crowded atmosphere versus a non-crowded atmosphere these metal ions will also play key role in folding the protein. So, inside the crowded environment of the cell this metal ion can play a very crucial role in folding the protein. This folded protein can give rise to the new conformation to the peptides and protein. As I mentioned metal ions can mediate protein folding which can have physiological and pathological consequences. Let us look at one interesting example of the calcium binding with a protein or which is known as calmodulin or calcium binding protein. So, this is the calcium binding protein, this is the apostate of cam or calcium modulating protein and this is the hollow cam. This calmodulin which is calcium modulating protein can consist of usually consists of 149 amino acid monomer and it is expressed in all eukaryotic cell. And overall if you look at overall up to 1 percent of total protein mass binds 4 calcium ions which is quite exciting 1 percent of all protein mass that we have all the protein that we have 1 percent of that binds with 4 calcium ion. The binding constant is quite good and the dissociation constant is of course, of course, not that high right. So, this overall calcium binding tendency to the cam or calcium modulating protein is going to give rise to the very order conformation. Let us say without calcium it was completely disorganized with calcium binding as you can see it gets organized in a particular fashion right. So, calcium to plus on the apostate of calcium modulating protein can gives rise to a open hollow cam structure. This is a closed structure this is not useful for lot of function, but in presence of calcium we have a very highly structured situation which is essentially can do quite exciting chemistry and the biological function. Now, we can try to look at these domain separately and try to see what is there in this organized structure. Where is the calcium? Calcium 2 calcium are here 2 calcium are there. Now, these are EF hand domain and these are having many crystal structure and you know first crystal structure was obtained with parvalbumin. Now, this helix loop again helix this structure is quite interesting and quite unique for the calcium binding and the calcium this cam protein right. This helix loop and the helix structure is quite interesting and we can if we can zoom into that we find that there are 9 residue into it and EF hand domains almost always occurs in pairs. Let us look at some of the more closer structure of it. So, this is helix E right and this is helix F that was in the yellow and the blue fragment and as you have seen the calcium is binding over here. So, the helix loop helix structure this helix loop helix structure are quite essentially a very organized structure that is controlled by the calcium binding. This EF hand domain almost always occurs in pairs. As you can clearly see the calcium is bound in this loop you can see in the right hand motif this is the helix E, this is helix F and the calcium binding happening over there. If you zoom on to this site more carefully the calcium binding with the protein backbone can be seen very explicitly. The primary coordination sphere of calcium modulating protein cam is looking like this it has 7 coordinated calcium site. So, this calcium is 7 coordinated we have aspartate, we have main chain and we have another aspartate, we have aspartate this is protein main chain, this is glutamate, this is water molecule, water aspartate, main chain aspartate, glutamate and aspartate. Overall this gives rise to a situation where the primary coordination sphere of calcium is, calcium is 7 coordinated and this is the crystal structure very clearly showing in how in calmodulin we have a clear calcium binding. Well there are many factors that influence calcium coordination to EF hand domain. One of them is certainly the cooperativity and another interesting issue is the cellular magnesium that also controls the calcium coordination to EF hand domain. After binding with calcium this cam protein now is ready to bind with different cellular targets and can promote exciting chemistry. Today we will stop here and in the next class we will show how calcium bound protein that is cam calcium modulating protein when it is bound with calcium can affect a series of events and that is why perhaps it is called as a messenger protein. It sends the message to do a particular activity or it activates a particular protein by binding with calcium in the cam right. Of course, KD values of EF hand domains of calcium and vary usually from 10 to the power minus 9 to 10 to the power minus 4 molar and there are many clear correlation between calcium affinity and variation in EF hand domains let us not discuss too much into that, but overall what we have seen that the calcium in particular in the last part of this class can bind with the protein and these are found a lot in our body. Overall 1 percent of the protein mass binds with 4 per 4 calcium center that is quite phenomenal and how it is bound or how it is binding is quite exciting as you have seen the EF hand domain motif where calcium is really bound tightly in the helix loop helix fashion and the coordination environment is quite crystal clear and has 7 coordination with the calcium. The effect of this calcium binding to cam and how it triggers a series of events will be discussed in that next class. Please keep studying, we will come back soon. Thank you very much.