hey everybody welcome back to general biochemistry lecture this time we're covering chapter three from lenninger principles of biochemistry this chapter covers amino acids peptides and proteins and i'm not gonna lie it's one of my favorite chapters these are the key principles that you should be able to understand and expand upon by the end of this lecture so make sure that you go back to this slide and look at these four principles make sure that they make sense we'll start with talking about amino acids going to do a little bit of a review of isomers because you need to know some terminology an isomer is a compound that has this they have the same chemical formula but different properties and from there you can branch off to talk about structural isomers which have different bonds we're not talking about those we're talking about stereoisomers which have the same bonds but different spatial arrangements and within that category you can have diastereomers where you're talking about cis versus trans arrangements or enantiomers and those are the mirror images that's what we're going to be talking about today when we talk about amino acids enantiomers so just a heads up if you don't remember that from general chemistry slash organic chemistry you may want to go back and review that we're not going to be too heavy in labeling things as diastereomers versus enantiomers or anything like that but you want to make sure that you understand what an enantiomer is and what that means in terms of amino acids so this is the generic structure for an amino acid there is an alpha carbon at the center and that alpha carbon is the chiral center all all amino acids except for glycine have a chiral carbon at the center the shape is tetrahedral and they all have this amino group here and this carboxylic acid there's a hydrogen and then there's this r group which is different depending on the side chain the side chain is what gives each amino acid its own little flare okay so we just went over all of these the carboxyl group amino group hydrogen and then the r group which is the side chain there are actually two possible stereoisomers for amino acids and they are enantiomers which means that they are a mirror image of each other so if you were to have a mirror right here what you would reflect is if you're looking if you have the l-alanine let's say looking in the mirror what you would see is d-alanine and this matters because the spatial arrangement means different things in terms of binding optically active that just means that if you had two separate mixtures one of l-alanine pure l-alanine the other one of pure d-alanine let's say since we have alanine on the slide here then you would see different um you'd see differences in terms of how those two compounds interact with light so all this means is that these enantiomers interact with light differently and you can detect that not going heavy into that either i just want you to know that that's what optically active means when you're looking at amino acids there's the d and the l and that tells you what configuration amino acid you're looking at for our purposes we're focused on the l amino acids because that's what you see in proteins there are five main classes of amino acids nonpolar aliphatic aromatic polar uncharged positively charged negatively charged you may be used to seeing fewer classes sometimes these two groups are lumped together to just say hydrophobic and these groups are lumped together to just stay charged but we're going to have a little bit more detail in this class let's start with the nonpolar aliphatic groups right so like we said before all amino acids have the same amino group carboxyl group and hydrogen but what's highlighted in this coral color is the r group and that's what gives them their specific properties aliphatic simply means like a carbon chain will be a little more specific hydrocarbon chain now there's one exception here there's a sulfur in this methionine but it's still largely it's an aliphatic group okay so all of these are nonpolar in nature and the hydrophobic effect that we talked about in chapter two these amino acids help stabilize protein structure through that hydrophobic effect they will cluster together and become stable and sequester themselves away from water you will need to memorize your amino acids for this class you'll need to be able to recognize them and draw them you'll also need to be able to draw short peptides as well and we'll get to that in a second the next group is aromatic r groups so these are the ones that have some kind of they've got double bonds you see can you see this uh benzene structure as a part of all of them they absorb light somewhere in the 270 to 280 nanometer range and when you're looking for protein concentrations and you're looking at that a particular wavelength of light around 280 you're looking at these amino acids now all proteins don't have a high concentration of phenylalanine tyrosine or tryptophan so if they have kind of reduced numbers of these particular residues you're not really going to see it that well that's just if you're in the lab tip these groups can also contribute to the hydrophobic effect just like the nonpolar aliphatic groups then we've got the polar uncharged r groups so they can form hydrogen bonds cysteine is special because it can form disulfide bonds so it will make bonds with other cysteine residues and form these disulfide bonds which you may also see termed disulfide bridges and those are also very important for protein structure we've got positively charged r groups they have a significant positive charge at a ph of seven and remember from chapter two ph sevenish is pretty biologically relevant so the charge around this ph is definitely relevant to protein structure and function then we've got the negatively charged r groups and they have a net negative charge at ph 7. again you'll need to know these so make sure that you recognize the structures and that you know the names there's also some uncommon amino acids that have important functions you don't need to memorize this this is just for your own edification so some common ones are 4-hydroxyproline and that is a modification that happens after protein is synthesized and is found in collagen then sometimes you have modifications that occur during protein synthesis such as pyrolysin another one we've got modified transiently to change a protein's function like phosphorylation we're going to talk a lot about those especially when it comes to metabolism and then we have some free metabolites like ornithine it's an intermediate in arginine biosynthesis sometimes intermediates can act as part of feedback mechanisms to control metabolic processes so all of these are uncommon amino acid examples that actually have pretty big roles again you don't need to memorize this this is just for your knowledge amino acids can act as acids or bases so if you're a little bit shaky on chapter two with how weak acids and weak bases um you know we talked about equilibrium and pkas and things like that you'll definitely want to show yourself up on that and then revisit the amino acids all amino acids have a non-ionic form i'm going to break up my highlighter here and in this non-ionic form that means that there's no charge so nh2 cooh no charge there the zwitterion form we've gained a hydrogen here and we've lost a hydrogen here the zwitter ion occurs around neutral ph and what that means is that this form has a net zero charge so that's the zwitter ion net zero charge zwitter ions can act as an acid or a base if it's acting as an acid it's going to lose a hydrogen or donate a hydrogen so i'm just highlighting here we go from that nh3 to nh2 so we're donating a hydrogen zone zotero ions can act as a base by picking up an extra hydrogen i should write that on the other side so this acts as a proton acceptor and we go from a negative charge here to no charge it says what our ions are very important when we're looking at a titration curve like the ones we talked about in chapter 2 we can get a lot of information we can get the pka of each ionizing group from an amino acid so each amino acid has at least two ionizing groups the amino group and the carboxyl group right now we're looking at glycine and its titration curve its r group is just another hydrogen clearly not hydrolyzable it is connected to a carbon what this means is that since you have two ionizable groups there are two regions of buffering power so these blue rectangles show the two different buffering zones that glycine has because of these two ionizable groups you'll notice this pi which we didn't we haven't introduced pi yet pi is the isoelectric point the isoelectric point is the point where you have the ion the zwitter ion with the net zero charge so if we're looking at this and trying to figure out which species of glycine was present at very low ph you're going to have this form here at about 7 so our pi is 5.97 so somewhere around neutral ph you have that zwitter ion and then at high ph or basic solution you're going to have this last form we're going to walk through this for different types of amino acids and class i'll show you a couple more examples but understanding a titration curve and which species of amino acid is present is definitely something you'll need to do for this class we're not finished talking about titration curves but i want to give you the heads up now so in general when you're looking at only two ionizable groups like with glycine you start with the cation which is something that's positively charged zwitter ion zero charge and then anion which is something that's negatively charged make sure that you understand this we'll talk a little bit more about that isoelectric point if you don't have an ionizable side chain remember that r group then you can calculate the isoelectric point by taking the average of pk1 and pk2 and pk1 and pk2 it's another way of saying pka1 and pka 2. same thing when you have ph that's equal to the pi the net charge is zero your amino acid is not really going to be that soluble in water and it doesn't migrate in an electric field it's got no charge when the ph is greater than the pi you're going to have a net negative charge and when the ph is less than the pi is going to be a net positive charge these are definitely things you should know because i can ask you a question about different amino acids maybe that don't have ionizable side chains so the non-polar ones and ask you at ph x what do you expect this amino acid to be you know is it positively charged negatively charged zero charge so the pkas for the carboxyl group and the amino group on an amino acid are different from a carboxyl group on its own and amino groups on their own the normal pka for a carboxyl group is about 4.8 but it's lowered in this case because the amino group withdraws electrons from the carboxyl group and that lowers its pka on the flip side the amino group in amino acids actually has a lower pka as well because of the carboxyl group so normally the pka for an amino group is about 10.6 so because these two groups are in close proximity they affect each other's pka since amino acids have these ionizable groups they can act as buffers we've got two buffering regions the closer you are to pka the better the buffer you have if i gave you a titration curve you should be able to tell me where the buffering regions are and and what ph you wouldn't want to have a buffer so in this case you would not want to have if you're trying to make a buffer at say seven it's not going to work too well okay so you should be able to look and see okay these are my buffering regions these are the ph's i'd want to be close to about 2 and somewhere in the ballpark of 10. so remember those amino acids that have ionizable side chains like the negatively charged ones positively charged ones well those side chains have a pka value as well so now instead of just pk1 and pk2 we've got pk1 pk2 pk3 and they can act as buffers as well and they influence the pi of the amino acid so if you have an ionizable side chain you can't just calculate the pi like you would with one that only has two ionizable groups you can still do a titration but you're going to have three ionization steps instead of two so here are a couple of examples and these look a little different than the uh glycine titration and you can see that you've got all these different um the different pk so you can have pk1 pk2 pkr or one two three so in your book you'll see pkr i may use pk 1 2 and 3 but understand it's the same thing looking at these structures and being able to tell the net charge is going to be key because that will tell you whether or not you're looking at one of the pkas or if you're looking at the pi in the case of glutamate your first value is pk1 then the next value that you get is your pi when you're doing this titration and knowing the different ionizable states of the amino acid will help you identify which points you're coming across as you're doing your titration in the case of histidine the order of events is pk1 pkr and then you're looking at the pi that's where as litter ion is so you want to take note of that charge and again we're going to do some practice in class to be able to identify these things net charge drawing the structures that kind of thing that's amino acids now we're going to string them together to make peptides and proteins peptides are just chains of amino acids and they're formed by condensation of two amino acids so you've got the oh from a carboxyl group on one amino acid and a hydrogen from the amino group on a second amino acid you lose a water and you form this peptide bond this is an amide bond you can also break these bonds through hydrolysis so that is using water to cut this bond apart not exactly as easy to do on the bench top but enzymes can do this quite easily when we're talking about peptides you can talk about them by the number so a dipeptide is two amino acids tri means three so that's three amino acids an oligopeptide it's a short peptide it's got a few amino acids polypeptide means that you've got something bigger more than 10 kilodaltons and then proteins have thousands of amino acids so much bigger than 10 kilodaltons it's just a little terminology when it comes to numbering and naming we're going to start with the amino terminal end so notice this free amine here this free amino group that is the n terminal end the amino terminal is what that n is short for that is the beginning of the peptide then you have all your other residues which residue is just another way of saying amino acid and then you have the carboxyl terminal end thought i had a different slide for that apparently not so the carboxy terminal end is here and that's just that free carboxyl group it's abbreviated c terminal when it comes to naming peptides you can use the full amino acid names we're not going to do that ain't nobody got time for that but you will need to know the three letter code abbreviations and the one letter code abbreviations so when you're memorizing the structures of the amino acids you'll also need to know their three letter code and the one letter code the amino acid composition of proteins is very highly variable so this chart is just a partial looking at cytochrome c cytochrome c is a part of the citric acid cycle or excuse me the oxidative phosphorylation it's all kind of linked in the mitochondria we're going to talk about that when we talk about metabolism and you can find cytochrome c across different species we also have chymotrypsin in this table and we're looking at these two proteins and their composition you know how many alanines how many arginines how many asparagines all that wildly different highly variable that's all the slide says you can estimate the number of amino acid residues which i'm not really going to hold you responsible for that but it's just something that if you are interested in doing some kind of research this is one of those tricks that you can use to get an idea of how many residues a protein has so if the average molecular weight of an amino acid is approximately 128 daltons and when you take two amino acids and condense them to form a peptide bond you're removing a water which is 18 daltons so that means that on average you're going to have about 110 for each amino acid so if you take the molecular weight of your whole protein divided by 110 that'll give you roughly the number of residues in that protein again not going to hold you responsible for that now all proteins are made of amino acids some proteins also have chemical groups aside from amino acids so we've got conjugated proteins and they have a permanently associated chemical component emphasis on permanently so the part that is the non-amino acid part is called a prosthetic group so we've got plenty of examples so there's lipoproteins that have lipids glycoproteins that have carbohydrates you see that a lot in the immune system phosphoproteins they have phosphate groups that are attached hemoproteins so hemoglobin we're going to talk about hemoglobin it's got this iron porphyrin group which is like it's a iron sulfur cluster so lots of examples metalloproteins are very important and they require a metal for activity so you see a lot of iron and zinc magnesium very very popular in the cell magnesium so lots of examples of these types of proteins and that prosthetic group is essential for the proteins function here's my favorite part working with proteins so proteins can be separated and purified and we can use the different properties of our protein of interest to separate it away from everything else you can separate based on size charge binding properties and solubility in general when you're purifying a protein the first thing you're going to do is take your protein source whether it's a tissue or a microbial cell that you've over expressed your protein of interest in and you're going to break it open it's also called lysis this gives you a crude extract it's all the proteins from the cell or the tissue in solution including all the bajillion d proteins that you don't want the second step is fractionation and so you're separating proteins based on size or on charge lots of different things that you can do there you can salt out a protein where you can exploit the differences in solubility lots of ways that you can fractionate and we'll talk about that in just a second then the third step is dialysis and that will take away all the salt and other things that you don't want with your pure protein away from your protein of interest when you're storing proteins when you're using proteins high salt metal contaminants all sorts of things can interfere with the function of your protein so column chromatography is something that if you are into proteins or enzymes you may end up doing a lot of as a graduate student or if you choose to do research um like an md phd program if you're interested in pre-med so this is definitely something that if you're interested in it you could spend a lifetime learning about this so the first step with any column chromatography is you have a buffered solution which is called your mobile phase that migrates through some kind of porous solid material and that's called the solid phase and that porous solid material is going it can have different properties that you're going to exploit so it could be charged um it could have uh some kind of a ligand that your protein is attracted to and will bind to so that mobile phase is the buffer that has you know it's going to make your your solid phase ready to receive your protein okay so the next step is to take a buffered solution that has your protein and migrate that through the solid phase and hopefully your protein will interact with the resin or maybe it's the only thing that doesn't interact with the resin that's you know also a possibility and the properties of your protein are going to affect the migration as the proteins migrate through the column you can collect them using a fraction collector and you can detect using usually using uv vis so 280 nanometer light which we said will detect those aromatic r groups of amino acids you can use that to detect protein so different types of chromatography ion exchange very popular it separates based on the sign and magnitude of the net electric charge of your protein so remember proteins are made of amino acids and those amino acids all have you know ionizable components some of them have r chains that are ionizable all of that together gives the protein itself a charge at different phs so it may be positively charged negatively charged or neutral and you can calculate that and then exploit that to be able to isolate your protein away from everything else so ph and the concentration of salt will affect your protein's ability to bind to an ion exchange column you can use cation exchangers or anion exchangers cation exchanges which is what's pictured here you're going to have negatively charged resin and you're going to have positively charged protein that interacts with that resin with anion exchangers you've got positively charged resin and negatively charged protein and again you can achieve that by changing the ph or the salt concentration there's also size exclusion size exclusion does exactly that it will separate based on size you'll also see it called gel filtration same thing the large proteins are going to emerge first from the column and then smaller and smaller and smaller the large proteins can't really interact with the resin so they pretty much just go straight through they kind of take the bypass the express lane and go straight through but the side the smaller proteins are small enough to interact with the matrix and so they're interacting with the you know the components of your solid phase and it takes them time to travel through so the smaller the protein is the longer it takes to travel large proteins alert elute first and then smaller smaller smaller then you can have affinity chromatography and this is based on binding affinity so some proteins interact with a ligand and that's just a small molecule that it will bind to and it'll bind really strongly you can exploit that and have your protein bind to that ligand and everything else will just keep on going about its business you can also use um protein tags so you can express proteins with a tag that it wouldn't normally have out in nature and that tag can be attracted to some kind of ligand on a column so that is also a great way to purify protein you can use hplc high performance liquid chromatography and that uses high pressure pumps to move proteins down the column and that will improve your resolution as well so here we're just looking at the different steps in the procedure and how pure your sample is so in the beginning you've got this crude cellular extract and in that extract you have a ton of protein right lots of protein here but in terms of your specific activity whatever reaction your protein catalyzes pretty small because that activity is diluted over a pretty large volume as you continue to purify and fractionate your activity your specific activity increases because you're removing more and more contaminants until at the end your last step you've got a very small volume but that small volume is all your protein so your specific activity very high and that's the goal to have a small volume lots of specific activity from your protein which means that you've got a lot of your protein now we can't just blindly do a purification process and say okay we're done there's protein in these tubes i dialyze them and i'm ready to go to town you need to use some diagnostic tools to actually make sure that you have the protein you think you have one of the tests that you can do is looking at the size of the protein using electrophoresis this is a technique that you can use to visualize and characterize purified proteins so you can estimate the number of different proteins in a mixture which is kind of related to that degree of purity which is the next point you can identify the isoelectric point and you can also approximate the the molar weight or molecular weight of your protein so you're going to take your protein sample and use an electric field to migrate those proteins across a polyacrylamide gel the proteins migrate based on a charge to mass ratio and they're visualized using kumasi blue dye there are other ways that you can stain but kumasi blue is pretty standard and it's the easiest notice that in this first lane there are markers these are molecular weight markers and that is just a standard that you can purchase that has specific amounts of proteins with known molecular weights and you can compare how your protein runs to the molecular marker to get an idea of what your molecular weight is the next lane has uninduced cells and we're going to talk more about this in class but if you're over expressing a protein in bacteria or some other cells you can crack open those cells you can crack open those cells before you start over expression and take a look at what your protein composition is and then you can induce and all of a sudden you see this band of protein appear that is very likely your protein of interest then you lyse the cells and you have your crude extract again you see this large band here you do an ammonium sulfate precipitation anion exchange and all these other whatever other things you're going to do to purify and fractionate your protein and you end up with this beautiful looking band at the end and hopefully it's what you want when you're running a protein gel you are very likely going to be using sodium dodecal sulfate or sds it is a detergent it binds to proteins and it partially unfolds them so that you're not running um you're not running a native gel but you're running a denatured gel that means that the proteins are kind of relaxed and open and you're going to have a similar charge to mass ratio for all your proteins so that the only difference is well this protein is bigger than another protein so it's going to move more slowly than the smaller proteins so sds helps to separate proteins by their molecular weight because we're not taking into account the differences in the protein size and shape there are ways that you can run what's called a native gel and that takes the protein as it is and let's run through a gel but oftentimes that's for a specific reason and you're not usually using that when you're doing protein expression purification if you want to estimate the molecular weight of a protein you have to have some kind of molecular weight marker proteins and that's what's again in this first lane usually this is something that you purchase and you receive you know some literature along with your purchase that tells you what the proteins are and their size in daltons if you plot this the distance of the their migration on the gel versus the log of their molecular weight then you will get a standard curve and you can take a distance measurement of where your protein runs and then you can kind of have an idea of what the log of that molecular weight is and then from there you can figure out the molecular weight of your protein it's not perfect but it's definitely you know it gets you in the ballpark so unseparated proteins can be detected and quantified based on their function we were talking about the specific activity where there's a difference between specific activity and activity in general so if you're purifying an enzyme you can follow the activity of your enzyme to see to make sure that you're actually you know purifying your enzyme and that that activity is increasing over time so activity is the total enzyme units in the solution specific activity takes into account the amount of enzyme activity per milligram of total protein so that is the number that was in the chart a few slides ago that takes into account okay we've got all this protein we've got this much activity as you purify the number of milligrams of protein total protein goes down which means your specific activity should increase over time so over the course purification it should increase now we're moving on to the structure of proteins there's different levels of structure there's four levels primary structure which is just the amino acid sequence the secondary structure is looking at recurring structural patterns alpha helices beta sheets those are secondary structure tertiary structure we're talking about the 3d shape of the protein and quaternary structure is when we're looking at more than one subunit so if you've got multiple subunits that come together to form some kind of complex that would be quaternary structure the amino acid sequence informs the 3d structure and that structure is important for us for specific functions so if you know something about the function of the protein that you may be able to guess what its structure is if you know about other proteins with similar functions and the reverse is true as well if you find some unknown protein and you can figure out its structure then you may be able to guess what its function is most human proteins are polymorphic and that means that they have variations in their amino acid sequence but remember with the amino acids we have different classes of amino acids and you may be able to substitute one amino acid for another and still maintain the overall characteristics so you can have something that's aromatic well you've got a few choices for that you want something nonpolar and aliphatic there's a few choices for that charged there's several choices for that so doing those kinds of swaps it could be a net zero change in terms of the structure and function of the protein because proteins are so important to life in order to study them we got to be able to take them apart and edmund degradation is a classic method of sequencing amino acids other techniques for sequencing amino acids and big polypeptides have been developed based on this method traditionally when you're trying to figure out the sequence of a protein you've got two options you can label the protein and figure it out you can break the protein apart figure it out if you're going to use labeling there are lots of different chemicals that will label the amino terminal amino group so remember the first amino acid has that free amino group and that is available to do chemistry so you can modify with all kinds of different chemicals to label that group and from there you can do a whole host of things to isolate one peptide at a time and when it comes to edmund degradation you're going to first label and that is at higher ph then you can cleave that labeled protein or that labeled amino acid at low ph and taking advantage of the difference in ph means that you can have labeling complete 100 and then change the ph and have cleavage 100 and then from there you can identify the free amino acid and then start again label cleave identify the free amino acid and all of these labeling techniques are based on that kind of a premise you can also study protein structure by breaking bonds so you can break disulfide bonds and then see what happens you can denature proteins we're not really going to focus on that too much though one great way to study protein structure is using proteases proteases are enzymes that catalytically hydrolyze peptide bonds far more efficient than chemical means there's a whole host of different proteases you don't need to know any of them specifically but you should know that different proteases have different cleavage points so they recognize certain sequences of amino acids and they will reliably cleave proteins in those areas in a certain way and you can take that information and you can say digest a protein with two or three or five different proteases and use the overlapping sequences to help figure out the entire protein structure you can couple that with mass spectrometry so you can digest a protein and then use mass spectrometry which helps you to measure molecular mass with really high accuracy so you can sequence and you can actually look at the entire proteome which is all the proteins we're just going to talk about the general steps involved in mass spectrometry mass spec is something that you could again spend a lifetime on very interesting very complex a lot of math believe it or not these machines are like modern marvels in my mind and very powerful tool if you're interested in doing any kind of research especially like metabolic research we're looking at a lot of different metabolites mass spectrometry is something you'll definitely want to learn about the first step for mass spec is to ionize the analytes in a vacuum what that means is you're going to take your peptide pieces those are the analytes and you're going to give them a charge then you're going to introduce those charged molecules to an electric field or potentially a magnetic field there's lots of different mass spec there's lots of different ways to ionize and then introduce to a field there's different types of mass spectrometry so it really depends then those charged molecules move through the field as a function of their mass to charge ratio or the m over z and then after that you can deduce the mass of your analyte again we're not going to go into detail about how to do all those things we will talk about combining liquid chromatography which is what we talked about with all the affinity columns and the ion exchange and things like that you can use liquid chromatography along with mass spectrometry and you can link up different types of mass spectrometry so you can do what's called lc tandem ms you can have mixtures of peptides and that's how we get to kind of looking at the whole proteome resolve those peptides that you first use chromatography to kind of separate and and then look at mass back and then you can identify proteins and protein abundance very cool stuff not gonna bore you with it but if you're interested let me know now we talked a lot about figuring out this you know sequence of a protein you can actually make proteins too so if you wanted to make a small peptide or a small protein you can do that chemically we're not going to talk specifically about the method even though it is shown here on the right we're just going to talk about a couple of the main points in terms of how you do this you have one end of your peptide that's attached to resin on a column and you protect the groups that you don't want to be chemically modified usually with f mock okay don't worry about what that is just know that it is a it's a protectant you can use whatever chemistry you need to to add on your next protein or excuse me your next amino acid and then you block that and then you continue over and over again until you have made your peptide or your protein chemically it's not exactly the most efficient enzymes are much better at this than we are in the lab but if you need a small peptide or something for what you're doing in the lab it can be an alternative to purchasing a peptide there are actually um automated machines that will do this for you versus you having to do all of the steps yourself and be your own organic chemist so that's all we're going to say here about synthesizing your own proteins chemically so a couple more notes on amino acid sequence the sequence of a protein can provide biochemical information we already talked about how it can inform the structure so the tertiary structure and the function but we can also get information about the cellular location so maybe there is a tag or a sequence at the beginning or the end of the protein that all proteins with that tag are localized to the endoplasmic reticulum or there's some kind of prosthetic group that we know binds to this particular region and that prosthetic group maybe localizes it to the cellular membrane we can also look at evolution so the really important residues in a protein or an enzyme are typically conserved even when you look at different species you can look at oh so these residues have not changed these other ones are variable but at these points we typically see this amino acid residue here so on the right what we're looking at is kind of these conservation charts where the size of the letter indicates how well it's conserved and then n to c we're talking about the n-terminus to the c-terminus so really big letters conserved so in b that d which is aspartate highly conserved however there's some variation here so there's some conservation but not as much there's it's variable there so this is also called a consensus sequence you're looking at the most common amino acid at each of these positions when you do that you can actually help identify the functional segments of new proteins or unknown proteins and you can establish sequence and structural relationships so if we know that you know this particular consensus sequence is associated with this type of enzyme activity then we can start to establish different families and things of that nature we can also identify what's called horizontal gene transfer and that's when a gene or group of genes is transferred from one organism to another when you're trying to look at evolution these are probably not the genes that you want to look at and like i said you can use the evolution and look at these consensus sequences to start defining members of a protein family here's a little bit of terminology here and we're not going to focus too strongly on defining families and things of that nature but i do want you to have an understanding of how it's done and why it's important so here's some terms here for you homologs are homologous proteins and they're all just members of a protein family paralogs are homologues within the same species so that would be for example in humans you know we can we have different homologues ortholog would mean a human protein and say a cow protein and they're members of the same protein family but humans and cows are two different species so those proteins would be orthologs you can compare the protein sequences using a database and identify all of the similarities that would classify these proteins as members of the same family and we'll do a little bit of that in class to look at some examples and do some sleuthing ourselves so that's it for this one that was amino acids peptides and proteins thanks for watching make sure that you tune into live lecture where we're going to do some practice and we're going to expand on some of the more complex topics to give you some hands-on experience with working with them to prepare you for the exams and assignments that will be coming later on have a good one stay safe