all right welcome back biochemistry class to chemistry with reef so today we are going to continue our discussion of chapter four which is talking about protein structure and function and um and we're going to continue by talking about the awesome topic of protein denaturation uh and so we talk about protein denaturation because really what we're talking about is protein stability we're asking ourselves how stable is my stability how stable is my protein okay and so there are several ways that we can denature proteins what we call denature proteins and so one of the classic ways illustrated here in this slide is temperature and so one of the classic ways of measuring uh protein stability is to measure what we call a tm tn is a melting it's the melting temperature we call it that yes it's the same just like your chemistry however we're not talking about the transition between a solid and a liquid here we're talking about the transition between folded to unfolded okay so melting temperature here has a slightly different meaning but that is where it comes from it's still tm okay and so we know that one of the classic ways to unfold proteins is heat right there's a reason um that basically there's a reason that we don't want to get very hot there's a reason that biologically we regulate our temperature right and why we keep our temperature regulated because if we get too hot our proteins will unfold when we die okay that's why fevers are bad so one of the ways of comparing proteins and their stability is to talk about their melting point that's just a little up to tm right and so we have two examples here we have ribonuclease a and we have apo myoglobin now the april myoglobin we just want to be aware this is myoglobin apo means lacking without and so this is without without the prosthetic group prosthetic group that's what apo means so without prosthetic group in that case it's a heme group we're going to talk about that next chapter in a lot more detail so don't worry about that too much now but that's what april myoglobin is by the way it's not a separate protein it's myoglobin without the heme group okay and so what you compare is we can see that we can ask and i would ask you if we were in class right now i would ask you which one of these is more stable what do you think is better stability do you want a higher melting point or a lower melting point for greater stability that's right the answer is higher the protein with a higher melting point is the more stable protein right and so how we do this we run what's called a melting point curve right it kind of looks like a titration curve where we're going to graph the percent of the maximum signal and that signal is typically what they typically do is you use some kind of probe right uh that measures the level of unfolded so here 100 means 100 unfolded and so at low temperatures you've got very little unfolded and as you rise you'll slowly get um you'll slowly get your protein to start to unfold now you want to be careful of the definition of tm so the tm is defined as the temperature at which the protein is 50 denatured okay 50 denatured that is the definition of tm and so you're looking you're going to find your 50 here and you're going to track it over okay that's the definition of denature and so uh you want to just be aware that the 50 denatured um is basically saying 50 of your amino acids are not in the three-dimensional conformation they should be in their quote native form as we would call them right and so denaturation typically so there's an average denaturation of the state of a standard protein is about 0.4 kilojoules per mole per amino acid so the longer that basically a lot of times you'll see that bigger proteins a lot of times are a little bit more stable right so yes if you had 100 amino acids that would be 40 kilojoules per mole to one fold it so yeah it takes a lot of energy hence the high temperature right uh for for a lot of these guys but notice of course there are nothings you're really going to survive boiling get too hot and a lot of things are dead right so it gets really exciting but you can measure overall stability right by measuring the tm now i want to take a minute and i want to talk about right what methods we can use to denature proteins now we obviously have presented our first one right which is temperature and and that's usually the most common one that we would note right so when you ask this question of classes people are the most common answer well tourist temperature absolutely right you would heat up a protein that protein will unfold right that's why we talked about when we ran sds page right in hit nudge nudge when we ran sds page gels we talked about we talked about the power um there it goes we talked about the power of sds to denature proteins right and one of the things we do with that is we heat it up right so can there are some conditions right to denature proteins d nature so conditions to denature proteins number one we have is heat absolutely watch out for heat right too hot and the proteins unfold you can also notice that a little bit rare is too cold can be bad for some proteins too by the way uh if you go too cold their interactions can get messed up as well but heat is usually the answer get too hot it's bad now what else can we change around so let's think about it right so what did what did we note when we were doing infinity chromatography what did we make a note of that we could change to make something elude off the column right so if it was reliant on charges or ion exchange chromatographies if it's reliant on charges right and having charges in very specific positions then what else could we potentially change to change the charges on the individual amino acids that are folded correctly or not well of course the ph oh it's not ready sorry the ph ph variations now the ph variations and what results in unfolding is entirely protein dependent every protein is different different proteins have different sensitivities to ph and they have different ph ranges where they work and cold and where they don't so it's highly dependent this is protein dependent right so protein dependent you just want to note that depends on your protein now what about that man that we've given us a big hint page gel right so whether we talk about what would we use to denature our protein we used sds well what did we say sds was does anyone remember so right aka the scientific version the scientific word we use for that is called detergents detergents like sds like not that like s the s the charges like sds will avoid the nature of proteins you basically put in groups uh you put in a molecule that has uh hydrophobic groups that suddenly the hydrophobic groups in the protein are suddenly attracted to the hydroponic groups in the sds they're like hey well water's excluding us let's all get excluded together and so they get excluded right and now your protein is unfolded because suddenly you've got those detergent molecules it's completely disrupting the hydrophobic interactions and so you've completely unfolded your protein your proteins hose right so you can use detergents now the fourth one is a little bit harder right detergents are referring to things like sds soaps right so detergent typically implies an am like right a very empty what we talk about we'll call this remember anthony pathetic molecules right so detergents are uh very a little bit specific right to like soaps so what if though i had a small molecule what if i used a small molecule to do something similar i want to disrupt those interactions the hydrophobic interactions the hydrogen bonding interactions i want to disrupt those but we have a name for those and they're called karyotropic agents stereotropic reagents okay carotropic meaning chaos inducing is how i think of it chaos tropic chaos making reagents right their job typically is to disrupt hydrophobic of the hydrophobic effect and sometimes h bonding right but mainly the hydrophobic effect and we'll talk about why that's so important the main example of a keratropic agent where you've got another slide don't worry is urea you may have heard of it so urea is used uh to disrupt to basically unfold the proteins uh it's also actually used to basically cut to mess with uh dna and rna as well by the way um it does the same kind of kind of thing but it disrupts those molecular interactions and it denatures them urea is the classic example of a chaotropic agent now you wouldn't note you should note with all of these that most of the time again guidelines instead of rules most of the time if you remove if you remove this view the effects so whatever they are your protein will refold most of the time so most of the time if you remove you can remove the detergent by the way it's really hard to remove detergents but a lot of times if you heat up a protein and you denature it if you cool it back down the protein will re-fold now now all the times will be filled correctly but it will refold same with ph with ph which is why one of the ways you gather off affinity columns is to vary the ph you break those interactions and then you re-establish the ph it'll re-establish the correct structure the correct um charge states of the amino acids and then refilled and go back to being proper so most of the time if you remove the effect right whatever it is whichever one of these conditions i should call this condition your protein will revolt and this is very very important okay so let's go back let's go back to our slides and i'm going to show you this is the these are the structures of some keotropic agents so here are two of them one of them is the guanidinium ion yeah that looks familiar that's the uh functional group on arginine rodinium ions let's take a look at these two different kaotropic agents take a look what do we see well we see one they're tiny as in they're gonna they're really small molecules that are gonna be able to navigate into the grooves and crevices of the proteins you'll also notice that they've got hydrogen bonding potential and in guanidinium ions shake case you have a charge and in the case of urea you basically see the again that size and the massive ability to hydrogen bond both donate hydrogen bonds and accept them right with that oxygen really fun stuff so again small they're going to go in they're going to disrupt those hydrophobic interactions right they're going to get in there and they really mess with the proteins so when you do protein denaturation you'll actually also note that you can do a tm based on a concentration of a keotropic agent it's a little bit rare but i've seen it in real papers where they'll basically note the tm and instead of being at temperature there'll be a concentration of a ketotropic agent like the guanidinium ion this is guanidinium hcl right and so you when you add concentrations notice that for this protein it took you know around 3.5 molar right gluten to reach the tm okay reach the fifty percent unfolded ferrari nucleus a it's pretty cool stuff all right uh so that'll bring us to a the idea of the hypothetical folding pathway we're going to talk about protein folding and so we're going to have going to go ahead and we're going to stop this video and take a break right here this is a good good time because we're going to talk about protein folding for our next video and it's going to be extremely super exciting all right thank you for hanging out on chemistry with reef i will catch you next time