we've talked about how proteins are made from amino acids which then fold into their secondary structure which are alah heles and beta sheets and then into their tertiary and quary structure now we're going to look at the structure and function of a particular type of protein and we're going to use hemoglobin and myoglobin as our examples why we use hemoglobin and myoglobin is because they are some of the first proteins that's whose structures we've known and so there's been a lot of studies on these proteins so let's take a look at these all right so let's go through the role of both myoglobin and hemoglobin all right so like we said they're the first protein structures that were ever determined and they are oxygen carriers so if we imagine an organism with lungs or gills oxygen is going to be coming in through those lungs or gills and carried in the arteries um if you have hemoglobin hemoglobin is your transport molecule for oxygen and it's going to transport the oxygen from the lungs to the tissues where the oxygen is needed hemoglobin that has oxygen bound is going to be called oxyhemoglobin when it gets to the tissues hemoglobin is going to release its oxygen and the deoxyhemoglobin or the hemoglobin without oxygen in it is then going to return to the lungs in order to get more oxygen all right so hemoglobin is a tetrameric protein here um so it's going to bind four oxygens at a time and um it is that transport molecule now in the tissues we're going to be using myoglobin myoglobin is going to be a storage pro protein mostly so it also has iron just like hemoglobin does and it's going to bind oxygen and then store the oxygen for later use if there's an oxygen deficiency oxygen can then uh be released from the myoglobin into the tissues itself all right so that is just an overview of how myoglobin and hemoglobin work generally let's go into a little bit more detail so here is myoglobin so myoglobin is a monomeric protein which means it stops at the tertiary structure it does not have a quinary structure we can see from its structure we have eight Alpha helices so we've got those colorcoded in here and in our protein is this heem prosthetic group so this heem is a small molecule and we can see it down here with carbons and nitrogens and it is going to bind oxygen so the oxygen is going to bind to this iron that is in the heem and remember that heem is the prosthetic group is not um coal bound to the myoglobin it is actually in the myoglobin using intermolecular forces right if we compare to hemoglobin hemoglobin is going to be a tetrameric protein so it's got four subunits we've got two alpha subunits and two beta subunits so two two that are one structure and two that are another structure but all four subunits are going to have a heem in it so this is a little bit complicated but we can see we've got an alpha subunit here and down here and then we've got a beta over here and over here I like this simplistic view because we can also see the heem groups and then the in pink and then these little circles within the heem are the irons if we look at the myoglobin and hemoglobin structure they're actually very similar uh so here we've got myoglobin and if we look at the shape that the myoglobin is making we can see that it overlays really nicely with this alpha 1 subunit of hemoglobin so they're very similar structurally and if we imagine taking this myoglobin and rotating it 180° it would fit right here on this U beta 2 subunit so they're very structurally similar all right let's take a deeper look at this heem group so again this heem is going to be non-covalently bonded into the protein so it's it's held in by intermolecular forces in the center we have this iron atom this iron 2+ and it is connected to the four nitrogens in the he so oxygen is going to be able to bind coal to the heem iron and that's going to be stabilized through hydrogen bonding with our histadine so we've got this histadine residue here this red bar here which we can see blown up here that's our O2 bound to our iron so that's our oxygen molecule this histadine here is hydrogen bonding with that oxygen in order to stabilize its presence in our um enzyme binding site on the other side of our oxygen so here's our O2 hydrogen bonding to that histadine on the other side is what we call the proximal histadine this histadine is binding to the iron so it's able to keep help keep that um that that heem in in place so this heem group is in a hydrophobic part of our he of our um myoglobin protein and our hemoglobin protein so here's just a little bit better view of what this looks like we've got our iron it has the four nitrogens of the parole ring that Tetra perole which is in he we've got the oxygen bound to the iron it's hydrogen bonding to this his residue this is on the the the um distal distal side and then we've got the histadine on the proximal side that is helping stabilize the heem in that hydrophobic pocket okay so let's look at the oxygen binding curves for both hemoglobin and myoglobin so for hemoglobin um this area here is the area of our tissues so what we have on our x-axis is partial pressure of oxygen and it's in tour TS the same as millimeters of mercury if you're not familiar with T so this would be a 10 uh millim of mercury of oxygen all the way up to 100 mm of oxygen on our y AIS we have a fraction of our protein that has oxygen attached so down here would be 0% of our protein having oxygen attached here would be 50% or5 of a fraction and then one one fraction or 100% of our protein has oxygen on it if we are examining our pink curve here we can see that when we have a partial pressure of 100 T of oxygen uh it is completely filled with O2 on our myoglobin as we are going down in partial pressure it's holding on to that oxygen fairly tightly and it doesn't get to what is called a a p50 which is where we have 50% of our protein with oxygen until a partial pressure of 2.8 T which is a very low partial pressure of o2 and then it will quickly drop from there to um to at zero um T of oxygen so what this means is that myoglobin is holding on very tightly to the oxygen until oxygen con concentrations become very low in comparison hemoglobin at at this 100 partial pressure 100 mm of mercury of o2 is completely saturated with oxygen but it has this sigmoidal curve so that means that once it gets down into a moderate concentration of o2 in this case between 20 to 40 mm of mercury of o2 then it will start to release the oxygen that's bound to it all right so this myoglobin again we has that hyperbolic O2 binding curve which means that the O2 is going to bind very tightly and will only release the O2 at very low partial pressures of o2 whereas the hemoglobin has this sigmoidal O2 binding curve so it kind of forms this s shape so it has a high Affinity at high concentrations and a low Affinity at low concentrations and releases at intermediate concentrations so let's talk about what that has to do with its function so remember that hemoglobin is going to be transporting oxygen so at a high concentration of o2 we want it to bind the oxygen then it's going to carry it to the tissues that are going to need the oxygen well the tissues that need the oxygen are going to have a lower partial pressure of oxygen so this will be the partial pressure of oxygen in the tissues once it gets to these tissues we need it to release the oxygen to the tissues so that's why it's important for hemoglobin to to release at a higher partial pressure of oxygen and now it has a lower partial pressure and it can go back to the lungs and pick up more oxygen in contrast myoglobin is not a transport protein it is a storage protein so we need it to store oxygen even through these higher these moderate concentrations of o2 that the tissues have we want it to only release O2 when we are in desperate need of o2 when our partial pressure of o2 is very low that's when we need myoglobin to release our O2 concentration so that's what how it works as a storage molecule is it only releases when we have this low concentration of oxygen so oxygen shows what we call positive cooperativity which means that when one subunit remember there's four subunits in hemoglobin when one subunit binds oxygen then all the rest are easier to bind oxygen so oxygen it increases as each O2 is molecule binds to hemoglobin and this is because of a confirmational change in the protein so we've got two different forms of hemoglobin we've got this deoxygenated form which is called the T or the tense form this t or tense form has a low affinity for oxygen whereas our oxygenated form has what is called a relaxed form a relaxed form of hemoglobin is going to have high Infinity so let's look at what this looks like so these uh four parts here represent the four subunits of hemoglobin and if it has a square corner like we see up here this is the low Affinity or t form of our hemoglobin if it has a rounded form which we can see here or here or here this is our R form our relaxed State this has a higher affinity for o oxygen so if there's no Oxygen bound which is what we see here all of these subunits are um are white that indicates that there's no Oxygen bound since they are all in white there's no Oxygen bound most of these are in their T form we can see that this subunit here seems to be sampling a confirmation of that R form um and that might make it it's a good idea for the hemoglobin to be able to sample these different confirmations um even though it's not as stable because that's actually how it may bind an oxygen if it comes upon an oxygen here we have a low oxygen concentration and what we see is that oxygen is not situated evenly distributed among all of the hemoglobin instead what we see is that this hemoglobin has two subunits with oxygen and this hemoglobin has two subunits with oxygen and that is because we have got this positive cooperativity if we did not have positive cooperativity what we would expect is that each hemoglobin might have one oxygen bound to it instead we see that one of them Bound in oxygen and that caused a confirmational change that allowed another subunit to bind another molecule of oxygen as we increase oxygen confirmation concentration now we can see that more subunits are getting oxygen bound to them and then as we reach our highest concentration of oxygen so that this may be like 90% um oxygen saturation we can see that we've got a lot of subunits that are fully oxygenated and some that are approaching that but what we're looking at is that oxygen only is binding in this R form this relaxed form and it is cooperative binding which means one subunit binding in oxygen will induce a confirmational change in another subunit that allows it to then also bind oxygen so here we have our partial pressure of o2 and our fraction of of subunits that have oxygen bound or fraction of um hemoglobin that has oxygen bound and we can see down here at the low partial pressures we have this deoxy form of hemoglobin and then as we are increasing oxygen we have more subunits with oxygen bound until we get to a high concentration of o2 in which case we will have hemoglobin fully oxygenated so we're going from our lower Affinity form to our higher Affinity form of hemoglobin we'll look here at the structural differences between the T confirmation and R confirmation so T is our tense confirmation and so we can see um that as we're looking at our tense confirmation versus our R confirmation there's a 15° shift in some of our subunits so here this blue subunit if you take a look at it here in the tense confirmation versus the relax confirmation we can see that it has moved or rotated 15° so take a look at this hee here versus here you can see it's more accessible in this relaxed or R confirmation um and because of this uh tea confirmation when you were in the deoxygenated form what will happen is that this iron in the heem uh is going to be pulled back by the histadine so over here the the iron is hidden or less exposed whereas when you're in the r confirmation that is going to make your heem no longer be so in our deoxygenated our heem is nonplanar when we go to our oxygenated form our heem is able to be planer exposing the iron and so this iron then is able to bind the O2 because it is not being hidden by the bent um the bent he here so this he this Helix that is right here is able to come and push the iron um down into the heem a little bit better in this oxygenated position and so when you are able to have one subunit that binds to oxygen it causes a confirmational shift in another subunit that now pushes that iron out so it's able to bind the oxygen more effectively okay this is called an aleric interaction so alisic interaction occurs when specific molecules reversibly bind to a protein and modulate the activity so in this case oxygen is our molecule that's binding to this protein and it's modulating the activity by making it bind oxygen and other subunits more effectively there are other aleric Regulators of um hemoglobin so we have 23 Vis phosphoglycerate or 23 BPG carbon dioxide and even protons so H+ are going to be alisic effectors of hemoglobin first need to look at how carbon dioxide can lead to a decrease in PH in our blood we have this um system that is in equilibrium where carbon dioxide will re react with water and that will form hydrogen carbonate and H+ ion so this again is an equilibrium so if you have high concentrations of carbon dioxide that's going to push the equilibrium towards having more H+ so remember that a decreased in your pH value means that you've got a higher concentration of H+ so as you have more carbon dioxide that's going to lead to more H+ which will cause a decrease in your pH all right I want to look at these green lines here which are our our hemoglobin binding curves so we've got hemoglobin binding at a pH of 7.6 hemoglobin binding at a pH of 7.2 and hemoglobin binding at a pH of 6.8 so if we're looking at our 6 7.2 this is what we would expect under physiological conditions so we've got a high concentration of o2 so our hemoglobin is going to bind our oxygen and then as we get to the tissues that need it which is in this Blue Area we're going to release that O2 and then we will um rapidly decrease as we have lower concent concentrations of o2 so again we're effectively releasing our O2 in our tissues now if we have a decrease in PH now we're going to be protonating some of the important residues that are key to getting your protein into its relaxed confirmation so that means that our protein is no longer able to get into its relaxed conf confirmation and so it's going to take on the T confirmation not the r confirmation that's that means that our protein here at this lower pH is going to be releasing its oxygen at a much higher partial pressure of o2 so it's going to be releasing the oxygen when it is not in the tissues so our O2 might be removed while it is traveling through the arteries for example and the oxygen does not get released in the tissues now if your pH is too basic like we see in the 7.6 it's going to resemble more like myoglobin and it is going to look more like a storage protein and it will not release the O2 until the concentration is far too low for O2 so we need to keep our ph balanced we need to keep it at this 7.2 in order to make sure that our hemoglobin is releasing the oxygen at the appropriate concentration which would be the concentration of o2 that is in our tissues okay BOS BOS bisphosphoglycerate so two3 bisphosphoglycerate is a small molecule and it is at the same concentration um as hemoglobin in adults in your body it is this very small molecule right here in this Center cavity and you can see it has a lot of negative charges on it and those are from the phosphate groups so if you bind this bis phosphoglycerate to hemoglobin it's going to cause a lower Affinity of for O2 so we can see that the amino acids in this cavity and these come from all of your different subunits have positive charges we can see some histadine we can see some lysines in here and so it's going to interact with your bisphosphoglycerate and what it's going to do is it's going to stabilize your tea confirmation so in that way bis phosphoglycerate is causing an alisic change to your protein that is going to regulate its activity it's going to make it more difficult for it to bind oxygen so a higher concentration of b phosphoglycerate means that you're going to bind oxygen less effectively interesting however is that fetal hemoglobin is going to have a lower affinity for this bis phosphoglycerate which means that when you have a an individual who is pregnant they are going to be able to they're going to be able to release biso phosphoglycerate in conditions of low oxygen and that low oxygen um level that high amount of bis phosphoglycerate will not affect the fetus and so the fetus will be getting oxygen first over the the mother um in the placenta while we're talking about hemoglobin it is really important that we pause and take a moment to talk about carbon monoxide poisoning and how it works so carbon monoxide when it binds to hemoglobin it binds with a greater Affinity than oxygen does uh and what it is going to do is it's going to bind and it's going to cause the hemoglobin to transition into its r State and bind oxygen which we think that should be a good thing we're binding oxygen however um what we see here here is our normal sigmoidal binding curve of hemoglobin in this case we call it carboxyhemoglobin but it's 0% because that means that there is no carbon monoxide or carboxy bound to your hemoglobin but if you were to have 60% carboxyhemoglobin so 60% of your hemoglobins have uh carbon monoxide on them that is going to change the binding curve of carbon monoxide and so you can see now it is more hyperbolic curve than a sigmoidal curve it is more similar to myog gloin in which case it is not going to release oxygen until you're at an exceptionally low concentration of o2 so your hemoglobin when it has carbon monoxide bound to it is not going to be able to release O2 unless your O2 concentrations are severely low carbon monoxide is not going to be considered an alisic effector because it is not irre it is not reversibly binding your protein it is instead irreversibly because of its high Affinity so what this is going to mean is that you're going to still have hemoglobin that binds oxygen just like it should when it goes into the lungs however it's not transporting the oxygen to the tissues or when it gets to the tissues it's not able to releasee its oxygen because now our oxygen has this higher affinity for ox our hemoglobin has a higher affinity for oxygen and so it's not able to release until your oxygen concentrations are very very low so you're going to have tissue death because those tissues are not going to get the oxygen that they need