hi everyone this is the video recording for uh week one section one video three and this video is on the chemistry of protein folding how the chemistry of the r groups and the amino acid drives how proteins fold basically so i'm going to go to our powerpoint slides okay laser pointer okay so as i said this section is on how the chemistry of the amino acids drives how proteins fold we'll talk about which bond types are involved at each level of protein structure we'll talk about which amino acids or which part of the amino acid makes those bonds the bottom line is that every protein has a unique amino acid sequence and remember that what's different in that amino acid sequence are the r groups right so the polypeptide backbone of every protein has the same chemical structure but what changes is the sequence of the r groups in that polypeptide and that unique sequence of r groups and their chemistry is what drives a protein to fold in its unique way so you as you think about that also think about what would happen if there are mutations that might change that amino acid sequence um and how that could change how the protein folds and that could change how the protein functions um so we'll we'll get into that a little bit and discuss that see oh the other thing i was going to add was that scientists molecular biologists and biochemists are not well what am i trying to say so protein folding is very complicated and the chemistry of it is very complicated and so we are not at a point where we can just take an amino acid sequence a polypeptide sequence and predict how a protein is going to fold um people are definitely trying to make um computer algorithms that can can try to predict that there are like competitions every year there's a lot of open source you know work or you know people do that for fun um but we are not at the point where um we could just feed an amino acid sequence into a computer and that computer could predict how that protein folds and so all the protein structures that you see are derived through other experimental methods which um i won't get into now but i will just say that they're very difficult and challenging and there are a lot of proteins for which we don't um have an atomic level sequence um but there are a lot of proteins that we do um so i just thought i'd mention that before we go in okay so let's start with primary structure right and so if you'll remember from the last video primary structure is defined as the sequence of amino acids in the polypeptide and so the polypeptide is held together by peptide bonds right so primary structure is held together by peptide bonds peptide bonds are covalent bonds all right and that's going to be important for some of our discussion about protein unfolding and misfolding a little bit i think in the next section because most of the sort of environmental factors like heat or salt or ph that we're going to talk about that cause proteins to unfold um those sorts of things at the the levels that we're talking about don't affect covalent bonds so can heat affect covalent bonds yes like right if it's like you know really really high heat but if you're talking about just like boiling temperatures um boiling temperatures do not affect the covalent bonds in the protein um however certainly could affect the non-covalent bonds so it's just a preview as to why i'm sort of pointing out specifically that peptide bonds are covalent bonds and how that can be relevant to protein structure and protein stability okay all right so then secondary structure and so in the last video um i define secondary structure as these really common folds that we see in proteins called alpha helices and beta pleated sheets or beta sheets okay and i didn't talk about what holds them together and so i'll talk about that now so secondary structure is held together by hydrogen bonds in the um hydrogen bonds formed between the backbone atoms meaning r groups do not make any direct interactions to form secondary structure okay that doesn't mean that our group chemistry can't sort of indirectly determine where secondary structure happens but our groups do not make any of the direct bonds that hold secondary structure together so those bonds are just hydrogen bonds and they're just between atoms in the backbone so specifically um if you look at backbone structure um which i think you can see here in your beta pleated sheet you can see here's a central carbon with a an arc coming off of it um and actually let's go to it uh let's go to this one um so here's your central carbon here's an amino ah sorry here's the r group um here's the c double bond o that carbonyl group um here's your peptide bond right here uh the nitrogen connected to the hydrogen okay so secondary structure is formed between the oxygen from that carbonyl group you can see one over here for example and the hydrogen from that nh group in the polypeptide backbone and that's going to be true for alpha helices is true for beta sheets um let's see so you can see that bond you can see the little dot dot dot that's how we often show um hydrogen bonds a little um spaced dot structure you can see one here you can see one here here's the c double bond o oxygen is forming hydrogen bond with a hydrogen now if it's been a while since you thought about hydrogen bonds and electronegativity and dipoles i'll just give a brief reminder here so covalent bonds can be polar or nonpolar okay and that's determined by the electronegativity of the two atoms in the covalent bond some atoms are more electronegative than others right so if you look at a periodic table you can see some numbers but the long and short of it is that we'll just talk mostly about carbon hydrogen nitrogen and oxygen and carbon and hydrogen have electronegativities that are in the sort of the 2.2 to the 2.5 range oxygen and nitrogen have electronegativities that are more in the 3.5 range so anytime you have a bond between a carbon and a nitrogen or a carbon and oxygen or a nitrogen and a hydrogen let's see or an oxygen in the hydrogen electronegativities are going to be pretty different by a factor of about one if you look at those numbers um and so what happens is that the shared electrons in that covalent bond are not shared evenly across the bond and so the um atom that attracts the electrons more readily which are the oxygen and the nitrogen um those electrons essentially spend more time around those atoms than they do around the other less electronegative atom so you have basically a partial charge distribution around the molecule because the electrons which are negatively charged like to hang out more with one atom than the other um and so you have um dipoles in those polar covalent bonds so in the carbon oxygen example here oxygen is more electronegative than carbon and so oxygen sort of um attracts those electrons more often because the electrons are like moving around right so electrons are spending more time around the oxygen that gives oxygen a negative dipole sort of like a partial negative charge in a way all right and here if you look at the nitrogen and the hydrogen the nitrogen here is more electronegative than the hydrogen and so the electrons spend more time around the nitrogen less time around the hydrogen and that gives the hydrogen a partial positive charge or a positive dipole the positive dipole and hydrogen the negative dipole and oxygen are attracted to each other and so they form something called a hydrogen bond okay so there's your reminder of your polar covalent bonds and why they're polar and why hydrogen bonds form we'll continue to talk about that chemistry as we go along we'll talk about it more when we talk about our group chemistry um let's see then what i want to say about alpha helices versus beta sheets is that in alpha helices um the hydrogen bonds form between the r groups sorry not the r groups the hydrogen bonds form between the backbone atoms and it's always um the atom uh from one amino acid and then atom from another amino acid four amino acids away so here it shows a carbon oxygen so here you have the oxygen forming a hydrogen bond with the hydrogen which is four amino acids away all right and so alpha helixes are always set up that way and so you have that happening all the way up the helix and that is what holds that structure together in beta sheets which you have happening is that you have polypeptide backbone um going in this path you can see in the picture and then curving around and coming back and so between these stretches of polypeptide here is where the hydrogen bonds happen all right so here's one stretch of polypeptide it curves around or does something and here's another stretch of polypeptide backbone and that's where the hydrogen bonds form and that's what holds the beta pleated sheets together in that structure let me just go back for a sec so um i would highly recommend watching movies 4.2 and 4.4 in the movies folder uh those movies are showing you the structures of alpha helices and beta sheets showing you where the hydrogen bonds are and all that so you probably find that helpful but i want to show you something else and point out a few other things i really love this picture i don't know where i found it off of the internet but here in in section a it just again shows you the structure of the alpha helix it shows you these little dashed lines to show you where the hydrogen bonds are that are holding this together they show the r groups as these little greenish balls sticking out here and so you'll notice again that um our groups are not making the interactions that hold um alpha helices together the other thing that i want to point out here in this side view and top view is i want you to see where the r groups are all right and i want you to specifically see that the r groups are sticking out away from the alpha helix okay they're not on the inside um they're not sticking you know towards each other on the inside um they're sticking out away from the alpha helix um notice you have your little modeling view here where you have some ribbon structure and you also have some ball and stick model for your um or maybe wire frame um for your r groups and then right next to it showing uh some space fill view and so this is what the um alpha helix actually looks like um like i think essentially for real although it's obviously it's not colored balls like that but it gives you a better sense of the of the surface shape here is your top down view through the center of that alpha helix again see how the r groups are arrayed around the outside of alpha helix the reason that i point that out is because i want you as you as you visualize proteins and you visualize how they're folding and you visualize how they're making interactions with other molecules i just want you to see that the alpha helices have these r groups how with uh sorry we have these structures with r groups sticking out um away from the alpha helix and so those r groups are available to make chemical interactions either with other r groups in the folded protein or with other molecules it is not unusual to have alpha helices uh be a major part of dna binding proteins where if you've ever seen the structure of dna there's there are groups in dna and that twisted ladder structure and the alpha helix is right the right size to fit into the major groove of dna and it's the r groups that stick out away from the alpha helix that make the specific contacts with the dna nucleotides so i just want to point that out that you have these surfaces that are going to be unique right you're going to have these unique r group sequences along the face of the alpha helix and so they can make unique interactions either within the folded protein or with other molecules okay and also beta sheets so i want you to notice that first of all beta pleated sheets are not completely flat they're kind of like twisted sheets but again our groups are arrayed above and below and the way it works is that you'll have one r group sticking up above and then the next adjacent are group six below and the next adjacent r group six above and then the next group six below so on and so forth and so again you have this situation where you could have really unique chemistry um on one side of the the beta sheet versus the other so if this were part of a folded protein you could have the outside of the folded protein which is going to be a more aqueous solution this is going to need to be more polar meaning you would have more polar covalent bonds in these r groups here whereas if this were facing the inside of the protein this might be more non-polar i realize that i'm getting a little bit ahead of myself i'm just hoping this is review for you and if it's not review for you then you know we're going to get to what polar and non-polar mean um so yeah and so i just want to point out that you can just have again unique chemistry interfaces on either side of the beta sheet because of where the r groups are in b and c this isn't super relevant to what we're talking about but it's just a nifty structure so these are called beta barrels um they are obviously proteins that form barrel shaped structures using alpha helices they're really common in poor proteins so proteins that sit in the cell membrane and allow things to pass through often regulated but they make great poor proteins in the in the cell membrane um right i think that's it for that slide okay um now i realize this video is going to be a little bit long so if you find that you need to take a break feel free to take a break i just wanted to include all of this in the same video because it's just all relevant to protein folding and chemistry okay so here you see the structures of all 20 amino acids and we are going to start talking about these because we're going to start talking about how the r groups derive overall folding of a polypeptide okay because the first two um levels of structure have been more general but the next two levels of structure are unique to every uh unique protein and our group uh sequence is what makes every protein unique um all right so you'll notice that the r groups or the amino acids are categorized into different categories you have the nonpolar category which we also call hydrophobic and then you have um just note that this is polar what we call uncharged and then here you have your charged categories we often call this polar charged and i make the point that these are all polar in green purple and blue polar meaning hydrophilic these are all amino acid r groups that are happy to interact with water and we'll talk about why and here your non-polar hydrophobic our groups tend not to like to interact with water the reason i bring this up is because hydrophobic versus hydrophilic chemistry so nonpolar versus polar is a major driver of protein folding and i can talk a little bit about why right now so i told you that electronegativity of atoms matters so you have your um bonds here notice in your r groups you have a lot of carbon-carbon bonds a lot of carbon-hydrogen bonds those are categorized as non-polar covalent bonds because the electronegativity between a carbon and a carbon is equal and then the electro negativity between a carbon and hydrogen is almost equal so they pre equally share their electrons and they don't have a partial charge distribution around that covalent bond that um that we would call that uh what am i trying to say so it's it's not the same as having unequal charge unequal electronegativity so on average the two atoms in a nonpolar covalent bond equally share the shared electrons okay and so because these equally share um their electrons pretty well around this r group it puts this in a nonpolar category now why do we call that hydrophobic the reason is because water is made up of oxygen bound to two hydrogens right and those are octu oxygen hydrogen bonds they're both polar covalent bonds um and so yeah they're both polar covalent bonds so water has a pretty good char partial charge distribution around the molecule molecules that have partial charge distributions like to interact with other molecules with partial charge distributions molecules without those strong partial charge distributions around the molecule like these nonpolar r groups really don't like interacting with other molecules with strong partial charge distributions so that is really important to cell chemistry in general and also really important to protein folding because the general rule is that non-polar r groups are going to want to interact with other non-polar r groups whereas your polar r groups your polar positively charged or polar negatively charged and your polar uncharged will be more comfortable making interactions with each other so when you have a polypeptide then you'll often have your non-polar r groups getting tucked away into the middle of the protein and making a very hydrophobic core because the surrounding area of the protein you know the solution that the protein is floating around in is a water-based solution right it's an aqueous solution in the cell um so this is this whole nonpolar versus polar is a major driver of protein folding um so we'll say more about that in just a little bit um let's see so then notice that you've got we'll go to we'll go to the polar uncharged notice in the polar uncharged r groups um you do have these polar covalent bonds so c o o h or polar covalent bonds here you have a c-o-o-h um here you have a carbon ring this is a benzene ring but you have an o-h on the end asparagine glutamine um you have you know your nitrogen and your oxygen which pulls pretty hard on the electrons and creates a partial charge distribution so these are all polar uncharged now why aren't they charged at the ph of the cell um well we won't get into that but that just has to do with their own unique chemistry so they tend not to be charged so they're still polar there's partial charge distributions around those molecules they can form hydrogen bonds right um but they are typically not charged at the ph um of the cell so the ph of the aqueous solution in the cell is typically about 7.4 um so you're positively charging your negatively charged r groups they do tend to be charged um at the ph of the cell now again they exist in dynamic equilibrium like we're talking right at the beginning of video one but do tend to spend quite a bit of time in their charge state especially lysine and arginine which have these amino groups that tend to stay positively charged and then your aspartic acid and your glutamic acid that have these carboxyl groups and tend to stay negatively charged good majority of the time histidine is kind of a special thing where it only spends about half the time positively charged it you know in an equilibrium state and so when we talk about positively charged r groups we're usually just talking about lysine and arginine histidine is certainly polar um it just doesn't come into the conversation as much for a positively charged r group um but it still has that kind of characteristic let's see there's a couple other um things i want to mention um one thing i want to mention as we get into the chemistry is just to explain and again i'll explain more about this later but to explain that like not all nonpolar r groups are equally non-polar hydrophobic so some are definitely more hydrophobic than others and so when we say that the more hydrophobic our groups are going to spend are really usually going to be tucked away in the middle of a folded protein you're really talking you're really hydrophobic r groups there are groups that are less hydrophobic so your glycine which only has a hydrogen as an r group that kind of doesn't have a lot of chemistry just in general um your alanine also is listed as non-polar hydrophobic not particularly hydrophobic um just because it's one methyl group um [Music] let's see your tryptophan because it has that one nitrogen even though it has a lot of carbon carbon carbon hydrogen bonds tends to pull that more into the less hydrophobic um category um let's see cystine uh fairly hydrophobic but in fact that sulfur hydrogen bond has a slightly polar flavor um although nothing compared to say like an oxygen hydrogen um finally uh this has nothing to do with hydrophobicity but i want you to notice proline and its structure proline is kind of a special case because its r group makes a cyclized um form with the backbone um so that gives proline a more rigid structure than um the rest of the r groups so again notice that its r group cycles back and actually makes a covalent bond with the nitrogen and the backbone um so that gives it a pretty rigid kind of kinked structure one of the things you'll actually find is that you won't find prolines in the middle of alpha helices because whatever this sort of kinked stable structure is it can't form into the alpha helix alpha helical structure of an alpha helix um you'll find them at the beginning and ending of alpha helices but you typically won't find them in the middle okay i think that's what i'm going to say for now we'll probably circle back to this diagram quite a bit all right i've said some of this already but we can always reiterate so nonpolar side chains are typically found tucked away in the middle of a folded protein and in fact this is a major driver of protein folding and actually holds the protein bound relatively stable stable e so it's a um something that really stabilizes the fold of a protein those hydrophobic r groups really don't like interacting with water and so they do everything they can pretty much to not interact with water which means they're really going to drive inwards to get away from the water as as the protein is folding so that's one thing to keep in mind so hydrophobic non-polar r groups in the middle polar r groups more around the outside of the folded protein and then here on the um right this is the right right anyways over here um what i i really like to show this diagram because what it basically shows is that it's not like you're either hydrophilic or hydrophobic um there's a whole continuum of really hydrophobic to really hydrophilic um the way they test this is to actually throw the pure amino acids into water and determine how much they precipitate or not um so soluble insoluble kind of um experiment so you'll notice that your charge r groups tend to be here and you're highly hydrophilic so your arginine lysine aspart aspartate which is the same thing as aspartic acid um glutamate which is the same thing as glutamic acid your asparagine and your glutamine and then your histidine so these are all really pretty hydrophilic r groups you've got your serine threonine prolene tyrosine tryptophan and cysteine sort of more in the middle and you should um work on your own to sort of um be able to describe and understand why those are more in the middle between your hydrophobic most hydrophobic and most hydrophilic right why is serine threonine tyrosine uh cysteine um i think tryptophan was in there yeah tryptophan you know why are those sort of more sitting on the fence as compared to some of the other ones and sort of be able to convince yourself of why that might be and even your proline i think the proline probably has a lot to do with the fact that it's connected to the nitrogen over here um and then you have your mo you're really hydro hydro you're really hydrophobic so isoleucine valine leucine phenylalanine methionine um so if you go back those are leucine isoleucine methionine phenylalanine valine those are going to be your most hydrophobic and you can see that they're all pretty much all carbon carbon carbon hydrogen bonds you do have a sulfur in your methionine but the difference in electronegativity between sulfur and carbon is is not a lot and so they're still sharing their electrons very equally and so you're not getting a lot of partial charge distribution around this molecule um and so it's considered you know fairly hydrophobic um your glycine in your alanine are here um sort of hydrophobic but not super hydrophobic um yeah and so when we talk about whether or not our group is going to be found in the middle or found on the outside just understand that you might actually find um some of the r groups that are labeled nonpolar you could still find them on the outside of a folded protein because they're not super hydrophobic okay so now we're going to get into the types of bonds that form in tertiary and quaternary structure again these are primarily r-group r-group interactions that's not to say that r groups don't interact with backbone or that backbone doesn't interact with backbone in tertiary structure you can get that but primarily what's holding tertiary structure and quaternary structure together are r-group r-group interactions and these are the general rules although there are certainly caveats to these rules because chemistry is complicated your polar uncharged sorry your polar charged r groups um your positive negative are going to form ionic bonds with one another right especially in their ionized states right you have one ion you have another ion they're attracted to each other because positive is attractive and negative right um i do note here that you can form electrostatic interactions with a polar uncharged r group as well so for example let's go back um you could have a positively charged nitrogen and lysine interacting with a negative dipole in the oxygen of asparagine um this isn't um this is not an ionic bond this is not a hydrogen bond it does have a name and i'm pretty sure it's just a dipole ion bond or something like that it doesn't really matter i just want to point out that that can certainly happen in these r group r group interactions okay now again these polar charge r groups are typically found on the outside of your protein your polar uncharged r groups are typically going to make hydrogen bonds with one another um they can make hydrogen bonds with one another or they can make hydrogen bonds with the backbone okay um and again we can go back to our structures so for example you could get this oxygen which has a negative dipole and glutamine forming um a hydrogen bond with this hydrogen bound to the nitrogen and asparagine because your oxygen is going to have a negative dipole your hydrogen is going to have a positive dipole and then they can happily interact with one another um and then also notice that you could certainly also have a situation where you have a hydrogen here an asparagine r group bound to the nitrogen so again the hydrogen is going to have an um positive dipole and you could have that making interaction with an oxygen bound to the carbon in this carbonyl group in the backbone right so something coming in and interacting with backbone okay so those are all hydrogen bonds that could that could form to hold proteins together now nonpolar r groups um are going to interact with one another primarily through something called hydrophobic effect and then van der waals interactions and again these are typically tucked away into the inside of the protein and what this means is that um and these these words are a little bit amorphous chemistry things but i'll just tell you that molecular biologists call this van der waals van der waals can be more of an umbrella term but i'll just say molecular biologists call these brand walls and what i'm talking about is that even when you have a carbon-carbon bond you have electrons moving between shared electrons moving the electrons are moving in between the two atoms and so over time on average they spend the same amount of time over the two atoms because they have equal electronegativities however that doesn't mean that at any given moment in time all the electrons are over on one atom or all the electrons are over on the other atom and so you can really form transient partial charges um but really really transient like really transient okay so transient partial charge distributions due to electron movement okay that's what we call van der waals so you they're very weak um they're the weakest of the charge interactions um but they can play certainly play a factor in um interactions that hydrophobic r groups are making with one another hydrophobic effect um is more of a i think of it as more of a thermodynamic idea that basically says that proteins are going to fold into their most stable state and so their most stable state means that hydrophobic r groups are not interacting with water you can do the same experiment with you know oils and oils and water right so oils are going to cluster together and try to exclude the water and the same thing happens with hydrophobic car groups in protein folding so the hydrophobic r groups are going to try to exclude the water or the polar r groups away and that behavior essentially is called hydrophobic effect there's more to it than that but we don't really need to go into that so we're just just trying to discuss the behavior of these molecules okay so those are the typical non-covalent bonds that are going to form between r groups and then sometimes between r groups and backbone that are going to hold a protein together these are all non-covalent they're mostly charged interactions if you'll notice that right if you don't know which i'm going to assume you do but i'm going to say it anyways um when you're doing charge interactions you know that negative and negative and positive and positive repel each other right and so um that can also be and have an effect on how proteins fold your two negative charges aren't going to want to get too close to each other two positive charges aren't going to want to get too close to each other whether those are dipoles or whether those are ions so that's just something to mention okay and then this is just a slide trying to show you um ionic bonds here between charged r groups and hydrogen bonds here it's showing a backbone backbone hydrogen bond um but you can certainly get hydrogen bonds between r groups or even between r group and backbone and then you have your van der waals with your clustering hydrophobic r groups here you have looks like valine uh alanine and baleen okay um everything that we talk about in terms of stabilizing tertiary structure is true for quaternary structure as well all right then there is one more kind of bond we have to talk about and this is actually a covalent bond that can help stabilize protein folding this is a covalent bond that forms between two r groups specifically cysteines and only cysteines two cysteines right cysteines have um a sulfi sulfhydryl group at the end the sh okay and there is a reaction that can occur in the cell that's catalyzed by um an enzyme that takes your two sh bonds of the two cysteines and forms a um does a this is an oxidation an oxidation reaction um where then there's a covalent bond formed between the two sulfurs and then the two hydrogens have to go away in the making of that again this is called a disulfide bond or a disulfide bridge let's see it's a covalent bond so it's the only covalent bond that actually holds tertiary structure or quaternary structure together it is not found in every protein it's not found in every um protein with tertiary structure it's not found in every protein quaternary structure um in quaternary structure what i'm saying is that this would be something that would hold the subunits together which we haven't talked about yet we'll get to that okay so um this is showing just a little cartoon of a polypeptide where your disulfide bonds would be holding things together um and here in your your little yellow bonds here in the folded protein now when you think about whether a protein should have a disulfide bond or not or should have several i want you to think about it from a structure function viewpoint there are proteins that when folded need to be a little bit more um i want to say loosey-goosey wobbly they need to have a little bit more wiggle room in their structure whereas there are proteins that need to be more rigidly folded or need to be more stable and so proteins that need to be more stable would be more likely to have a disulfide bond or a few disulfide bonds um and so that's just to think about it from a structure function relationship so not all proteins have disulfide bonds um but when they do they're really helpful for stabilizing their their fold their structure okay so quaternary structure as i said um same bonds are involved in holding the different polypeptides in um in quaternary structure together oh wait we did talk about subunits in the last video so i can talk about them okay so just i'll just remind you from the last video that in quaternary structure if a protein in its functional state is made up of more than one polypeptide then each of those polypeptides is called a subunit right and so we give i'm coming back to the example of hemoglobin and of antibodies okay so in hemoglobin remember you have four subunits two are alpha um subunits and two are beta subunits primarily alpha helical structures these guys are all held together so the different subunits are all held together by non-covalent bonds right there are no disulfide bonds in hemoglobins either at the tertiary level or at the quaternary level okay by tertiary level i mean that there are no disulfide bonds holding the tertiary structure together um and there are no disulfide bonds holding the subunits together okay however that's a little bit different in antibodies and now if you think about antibodies so if you know anything about antibodies they're often secreted outside the cell so they actually run around our body in our lymph system in our bloodstream or maybe just our lymph system i don't really know um but it's a much harsher environment if you're coming outside the cell so it probably makes a lot of sense that antibodies have lots of disulfide bonds plus antibodies can be quite rigid structures all they do is essentially act as sentinels or flags so they'll bind to an antigen something they see as foreign and then just sit there tagging that um molecule or particle or cell or whatever it is um and just waiting and attracting other cells to it so they don't have to change shape they uh need to be pretty stable because they're outside the cell they exist for long periods of time so they need to be pretty stable um so you find disulfide bonds in this protein and you you find disulfide bonds in the tertiary structure holding the subunit together as well as between subunits so in the quaternary structure as well so you can see here this is a light chain this is one of the heavy chains you can see a disulfide bond holding the light chain to the heavy chain and you can find disulfide bonds holding the two heavy chains together um and then with the other light chain and the other heavy chain okay so that's just to talk a little bit about why you might have disulfide bonds and why you might not okay um i think that is it for go protein structure okay so again i'm sorry if that was that video was a little bit long there was probably a lot in that video um again with any of these videos feel free to take notes feel free to pause the video you know you can always come back to it um and yeah and then there'll be practice questions and there'll be chances for you to ask questions if there's anything you're not sure about um you know links to the additional resources there's there's always access to all of that okay all right until next time next video