hello everybody my name is Iman welcome back to my YouTube channel today we're covering the last chapter for our MCAT biochem playlist this chapter is about non-enzymatic protein functions and protein analysis now typical functions provided by proteins within this cell include things like supporting cellular shape and organization and acting as enzymes now previously we've talked about enzymatic functions of proteins but we haven't focused too much on structural proteins or motor proteins or binding proteins and that's kind of one of the things that we want to start talking about in this chapter so we're going to start with first covering structural proteins now the primary structural proteins in the body are things like collagen elastin keratin actin and tubulin structural proteins generally have a very highly repaired additive secondary structure and a super secondary structure which is just a repetitive organization of secondary structural elements together that are referred to as a motive and this kind of regularity it really gives many structural proteins a fibria's nature so what we want to do is we just want to talk about a couple of these examples of structural proteins ultimately um these kinds of proteins are going to be proteins that play a role that in like composing the skeleton cytoskeleton and anchoring proteins and much of that extracellular Matrix collagen has a characteristic trihelical fiber and it makes up most of the extracellular Matrix of connective tissue it's really found throughout the body and it's important in providing things like strength and flexibility elastin is another important component of the extracellular Matrix of connective tissue its primary role is to stretch and then recoil like a spring which restores the original shape of the tissue keratins are actually intermediate filament proteins that will be found in epithelial cells they contribute to the mechanical Integrity of the cell and they also function as regulatory proteins keratin is the primary protein that will make up hair and that makes up hair and nails that we have acting actin is a protein that makes up microfilaments and the thin filaments in myofibrils it's actually one of the most abundant protein in eukaryotic cells actin proteins they have a positive and a negative side and this polarity actually allows motor proteins to travel unidirectional along an actin filament kind of like just a one-way Street and then last but not least in regards to structural proteins you should be familiar with is tubulin tubulin is a protein that makes up microtubules and microtubules are really important for providing structure for chromosome separation in mitosis and meiosis and intracellular transport within kinesin and dynein which are going to be motor proteins we talk about here in just a second um like actin tubulin also has polarity and the negative end of a microtubule is low-key is usually located near or adjacent to the nucleus whereas the positive end is usually in the periphery of a cell so that is structural proteins what about motor proteins now some structural proteins also have motor functions in the presence of motor proteins right the the the Cilia and flagella bacteria and and sperm are prime examples of this right because the contraction um are prime examples of this because motor proteins actually also display enzymatic activity and they aid in the in in the movement of structural proteins now um like we said monoproteins they display also enzymatic activity acting on ATP Aces that power the conformational change necessary for motor function and they are they have transient interactions with things like actin or micro or microtubules now myosin is the primary motor protein that interacts with actin in addition to its role as the thick filament in a myofibril myosin can also be involved in things like cellular transport each myosin subunit will have a single head and neck and movement at the neck is going to be responsible for the power stroke of a sarcomere contraction now um kinesicines and dinings written right here they are motor proteins that are generally associated with microtubules so myosin with actin and these guys with micro tubules and we said that tubulin is the protein that makes up microtubules by the way all right now these two are these two are motor proteins that are associated with microtubules they have two heads at least one of which remains attached to tubulin at all times Kinesis they play a role in aligning chromosomes during metaphase and depolymerizing micro tubules during anaphase of mitosis dienes are involved in the sliding movement of cilia and flagella both proteins are really important for vesicle transport in the cell the thing is that they have opposite polarity so kenosines they bring vesicles toward the positive end of microtubules and Dyne they bring vesicles towards the negative end um in neurons we can neurons are a good classic example of of the the consequences of these motor proteins polarities right because kinosines will bring vesicles of neurotransmitters to the positive end of an axonal microtubule so like towards the synaptic terminal whereas in contrast dynames will bring vesicles of waste or recycled nutrients neurotransmitters back towards that negative end of the microtubule toward the Soma right and so that's a really clear depiction of of some of the consequences of the polarity um the different opposite polarities of dynees and and Kines so in general as a short all right that's motor proteins they're responsible for muscle contraction and cellular movement fantastic then we also have binding proteins like well well Pro proteins they primarily exert enzymatic or structural functions within the cell they can also have you know some stabilizing functions in individual cells in the body proteins that act in this way will tend to transport or sequester molecules by binding to them and binding proteins including hemoglobin or calcium binding proteins or DNA binding proteins and others um they will act as some mean of of Transport or or sequestering molecules through that binding now each binding protein will have an affinity curve for its molecule of Interest um a transport protein which has to be able to bind or unbind to its Target to maintain steady state concentration that's likely to have varying Affinity depending on environmental conditions obviously all right so that's binding proteins for us what about cell adhesion molecules cell adhesion molecules also known as cams for short are proteins that are found on the surface of most cells and they aid in binding the cell to The extracellular Matrix or to other cells now while there are several different types of cams they are all essentially integral membrane proteins adhesion molecules they can be classified ultimately into three classes three major families or classes all right those are coherons integrins and selectins now we're going to just quickly briefly describe what each of these are CAD hereins are a group of glycoproteins that will mediate calcium-dependent cell adhesion all right they're often they often hold similar cell types together like epithelial cells um and different cells usually will have type specific adherence for example epithelial cells will use e cadherent while nerve cells are going to have you know their type specific cat hearings which are called ncat urine all right so that's cadherence integrands are a group of proteins that are going to have two membrane spanning chains called Alpha and beta and these chains are really important in binding to and communicating with the extracellular Matrix now integrins also play a really important role in cellular signaling they can also greatly impact cellular function by promoting cell division apoptosis or even other processes selectins selectins are unique because they bind to carbohydrate molecules that project from other cell surfaces these are bonds are the weakest formed by the by by cams they're expressed on white blood cells and the endothelial cells that line blood vessels and like integrins they also play a very important role in host defense including inflammation and white blood cell migration all right now in terms of maybe a little bit of a real world application many medications they'll Target selectins and integrands so for example research has shown that the the ability of cancer cells to metastasize or again break away from a tumor and then invade other distant tissues is associated with unique expression patterns of these cell adhesion molecules and then by targeting them by targeting these cell adhesion molecules metastasis may be avoided and then even to stop the clotting process maybe during heart attacks other medications will try to Target cell adhesion molecules used by platelets so those are some two real world medical applications of being able to understand cell adhesion molecules their role in different things and then being able to develop medication to prevent or support or whatever it may be to to help treat a specific illness or disease one last thing I want to cover under under the the umbrella of of talking about um cellular functions I want to talk about immunoglob globulins the immune system and it's very complex and it's made out of many types of cells and proteins and these cells and proteins they have a common purpose usually to get rid of the body to get rid of the to to rid the body of foreign Invaders now the most prominent type of protein that's found in the immune system is the antibody antibodies are also called immunoglobulins they are they are proteins that are produced by B cells that are going to function to neutralize Targets in the body so this word is just you know a synonym to antibody right antibodies are also referred to as immunoglobins and like we said they function to neutralize Targets in the body things like toxins and bacteria that shouldn't be there they also function then to recruit other cells to help eliminate the the thread now what we have here is is the structure of a antibody antibodies are usually kind of y-shaped right um that are made out of these two identical heavy chains all right and two of these lighter chains to identical lighter change uh chains then we have these disulfide links and non-covalent interactions that are going to hold the light and heavy chains together um and each antibody it has an antigen binding region at the tips of the Y alright so right here within this region there's going to be specific polypeptide sequences that will bind one and only one specific antigenic sequence the remaining part of the antibody molecule is then known as the constant region and it's involved in Recruitment and binding of other cells of the immune system things like microphages for example so when antibodies bind to their targets called antigens they can cause one of three outcomes all right one of three outcomes they can one neutralize the antigen making that pathogen or toxin unable to exert its effects on the body it can to Mark the pathogen for Destruction by other white blood cells immediately all right and this marking function is is also called opinization or three all right they can cause clumping together of the antigen and antibody into the large insoluble protein complexes that can be digested by microphages all right and so that's kind of a brief overview of cellular functions for for proteins besides just enzymatic functions right so we've kind of covered a good portion of this non-enzymatic protein function goal of this chapter right we've talked about structural proteins motor proteins binding proteins cell adhesion molecules and even antibodies now one thing I want to talk about before we get into the second main objective of this chapter which is talking about protein analysis I want to talk a little bit about bio signal biosignaling is a process in which cells receive and act on signals proteins participate in biosignaling in different capacities including acting as extracellular extracellular ligands or Transporters for facilitated diffusion or receptor proteins and and as second messengers right the proteins involved in BIO signaling can have functions in either substrate binding or even enzymatic activity now some things under this umbrella of biosyn mowing that we want to cover are things like ion channels um enzyme-linked receptors and G protein coupled receptors right because bio signaling can take advantage of of either existing gradients which would refer to ION channel channels or it can take advantage of second messenger Cascades which would fall under the guise of enzyme-linked receptors and NG protein-coupled receptors so that's why we want to cover these three we'll start with first ion channels ion channels are proteins that create specific Pathways for charged molecules all right they are classified into three main groups that have different mechanisms of opening but that all permit facilitated diffusion of charged particles all right if you remember facility facilitated diffusion is a type of passive transport it's the diffusion of molecules down a concentration gradient through a pore in the membrane that's created by some transmembrane protein it's used for molecules that are pretty much impermeable to the membrane all right so think you know back to a couple chapters ago that would be you know molecules that are large polar or uncharged now facilitated diffusion allows integral Pro membrane proteins to kind of serve as channels for these substrates so that they can avoid the hydrophobic fatty acid tails of the phospholipid bilayer the three main types of iron channels that you should know for the MCAT are ungated voltage-gated and ligand-gated all right so in ungated all right as the name suggests they have no Gates and they're therefore unregulated all right for example all cells possess ungated potassium channels that means there's going to be a net um efflux of potassium ions um through these channels unless potassium is at equilibrium all right then we have voltage gated channels this gate is regulated by the membrane potential change near the channel so for example many excitable cells like neurons they possess voltage-gated sodium channels those channels are closed under resting conditions but membrane depolarization can cause a protein conformation change that will then allow them to quickly open and then close as that voltage increases all right then there's also ligand-gated channels all right for these The Binding of a of a specific substance or ligand to the channel is going to cause it to either open or close for example neurotransmitters are going to act at ligand-gated channels at the postsynaptic membrane all right so that is going to be important for allowing in for the activity of of neurotransmitters all right so that's ion channeling that's ion channels all right we can also talk about enzyme-linked receptors so many you know membrane receptors they they can also display catalytic activity in response to ligand binding these enzyme-linked receptors they kind of have three primary protein domains they have a membrane spam spanning domain they have a ligand binding domain and they have a catalytic domain so the membrane spanning domain it's going to Anchor the receptor in the cell membrane the ligand binding domain is going to be stimulated by the appropriate ligand and that is going to induce a conformational change that will then activate the catalytic domain all right this often results in the initiation of some second messenger Cascade and a good example of this is receptor tyrosine kinases all right these are composed of a monomer that dimerizes upon ligand binding fantastic all right then there's also G protein coupled receptors G protein coupled receptors there are they are a large family of integral membrane proteins involved in Signal transduction now in they're they're usually characterized by their seven membrane spanning Alpha helices they The receptors differ in specificity of the like and binding area found on that extracellular surface of the cell and in order for these G protein-coupled receptors to transmit signals to an effector in the cell they utilize a heterotrimeric g protein G proteins are named for their intracellular ring link to guanine nucleotides so GDP and GTP and The Binding of a ligand is going to increase the Affinity of the receptor for the G protein now there are three main types of G proteins you should know okay we have Gs which stimulates the adenylate cyclase it's going to increase levels of camp in the cell all right then you have G1 which inhibits um adenylate cyclase and that's going to decrease levels of camp in the cell so this one increases CMP levels this one decreases CM Camp levels and then our third type is GQ GQ activates phospholipase C which Cleaves a phospholipid from the membrane to form pip2 so this one forms pip two pip2 can then be cleaved further into other molecules like dag and ip3 and ip3 specifically can open calcium channels in the endoplasmic reticulum thereby increasing calcium levels in the cell so these are the three G proteins you should probably be familiar with and their effects fantastic now what we can move into is protein isolation in order to better understand a specific protein it's going to be important to be able to isolate the protein for study the purification of proteins can be pretty much considered an art form when you consider the difficulty of of doing purification of proteins um and the difficulty of isolating just one protein from a cell containing hundreds to thousands of them now the most common isolation techniques and the ones that we have to worry about for the MCAT are electrophoresis and chromatography either of which can be used for Native or denatured proteins we'll first talk about electrophoresis electrophoresis is like we said one method of separating proteins it works by subjecting compounds to an electric field which moves um which moves those compounds according to their net charge and size so negatively charged compounds will migrate towards the positively charged electroid so negatively charged compounds will move towards that positive side positively charged um compounds will want to say closer to the negative electrode all right so negatively charged compounds will migrate towards the positively charged anoid and positively charged compounds are going to migrate towards the negatively charged anion now this the velocity of this migration is known as the migration velocity and we can actually write the the migration velocity as an equation all right where V is directly proportional to the electric field strength all right and to the net charge of the molecule which are we're going to denote as Z so E's electric field Z is charge all right and it's inversely proportional to the frictional coefficient which we denote F and this depends on the mass and the shape of the migrating molecules so now we have this equation for migration velocity that's directly proportional to electric field and charge inversely proportional to friction coefficient now these standard medium for protein electrophoresis is a polyacrum acrylamide gel this gel is going to be slightly porous it's a porous Matrix mixture and proteins travel through this Matrix in relation obviously to their size and charge the gel kind of acts like a sieve that allows smaller particles to pass through easily while kind of retaining larger particles so it's not just charge that will affect remember migration and migration velocity and Direction but also size okay so that's an important thing to keep in mind the molecule will move faster through the medium if it's smaller highly charged or placed in a large electric field and conversing molecules will migrate slower or not at all when they are bigger more convoluted electrically neutral neutral or placed in a very small electric field now there are different kinds of variations of electrophoresis that we're going to talk about and compare and contrast that's going to be native page and SDS page native page um is is this all right so polyacrylamide gel electrophoresis page is a method for analyzing proteins in their native States so that's what Native page is unfortunately page is limited by the varying Mass to charge and the mass to size ratios of cellular proteins because multiple different proteins may experience the same level of migration so in this native page electrophoresis the functional native protein it can be recovered from the gel after electrophoresis but only if the gel hasn't been stained because most stains will denature proteins Page's most useful to compare the molecular size or the charge of proteins known to be similar in size um but if that's not the case another useful technique is the SDS page method so SDS stands for sodium dodecile sulfate this polyacrylamide gel electrophoresis is is a useful tool because it separates proteins on the basis of mass alone so this technique starts with the premise of page but then it adds SDS which is a dis which is a detergent that will disrupt all non-covalent interactions it binds to proteins and it creates large chains with negative net negative charges and so it neutralizes the protein's originals charge and denatures the protein and so then as the protein moves to the the gel the only variable that's affecting their velocity is going to be the electric field strength and the frictional coefficient which depends on Mass so this is a good technique if you're only really interested in um you know it is useful when you're only separating proteins on the basis of mass alone now something to also keep in mind when you're talking about electrophoresis is that proteins can be separated on the basis of their isoelectric point all right pi and we've talked about this before too the pi is the pH at which the protein or amino acid is electrically neutral with so that means within that it has an equal number of positive and negative charges for individual amino acids this electrically neutral form is called the zwitterion if you remember and that is when the amino group is protonated the carboxyl group is deprotonated and the side chain is electrically neutral isoelectric focusing exploits the acidic and basic properties of amino acids by separating on the basis of that isoelectric point Pi so in short isoelectric focusing in in isoelectric focusing a protein will stop moving when the pH is equal to the p i all right fantastic so that's electrophoresis something else that we want to talk about when we talk about protein analysis protein separation um is chromatography chromatography is another tool that will use physical and chemical properties to separate and identify compounds from a complex mixture it refers to a variety of techniques that are going to require the homogenized protein mixture to be fractionated to be fractionated through a porous Matrix one of the reasons that this is a valuable tool is that the isolated protein is pretty much immediately available for identification and quantification all right now the the couple of chromatography techniques we're going to talk about here are all of these forms that we're going to talk about the concept is identical the more similar the compound is to the surroundings whether that's by polarity charge so on the more it will stick to and move slowly through its surroundings so remember that this is very important the more similar the compound is to its surrounding the more it will stick to its surrounding and that means the more slowly it will work through the surrounding all right chromatography is preferred over electrophoresis when you have large amounts of proteins that are being separated and and the process begins by placing the sample into a it begins by placing the sample into a solid medium called the stationary phase that's filled up in your little chromatography column all right or the absorbent the next step is after you run the sample through this is to run the mobile phase through the stationary phase this is going to allow the sample to run through the stationary phase or a loop and then depending on the relative Affinity of that sample for the stationary and mobile phase different substances will obviously migrate through this column at different speeds all right and that means you're going to be able to separate the different components in your sample all right components that have high affinity for the stationary phase will barely migrate at all components with high affinity for the mobile phase will obviously move much more quickly all right the amount of time that a compound can probably can spend in the stationary phase is referred to as the retention time varying retention times of each compound in the solution is gonna result in separation of components within the stationary phase or deep partitioning or partitioning all right so that means each component can be isolated individually for study and the only thing that you're thinking about is how fast each component will separate depending on if it's more attracted or similar to your stationary phase versus your mobile phase all right so you're thinking in terms of your stationary station oop phase versus your mobile phase all right usually if you have a column you pack it with like silica that's your stationary phase and then you pass through it some sort of liquid solvent that's your mobile phase if the sample that's passed through here is like your stationary phase then it's going to move slowly if it's like your mobile phase it will move more quickly all right and then based off of that you'll separate the different components of your sample um as as you as it passes through the column now we're going to talk about a couple of different kinds of chromatography we'll talk about column chromatography all right and column chromatography this is what I just displayed here a column is filled with silica or or aluminum beads as an absorbent and gravity moves the solvent and compounds down the column as the solution flows through that column both sides and polarity are going to have a role in determining how quickly a compound moves through the polar silica or alumina beads the less polar the compound the faster it can elude through the column so that means it's going to have a short retention time all right in column chromatography the solvent polarity pH or salinity can easily be changed to help elude the protein of interest and eventually the solvent the solvent will drip out of the end of the column and different fractions that leave the column are going to be collected over time all right I know I used column chromatography to to convey this idea right here which we discussed earlier but you have to remember that in column chromatography there are several things that are going to affect how fast something moves through said column things like solvent polarity pH salinity all those things are going to have a role all right so that's one kind of chromatography calling chromatography then we have ion exchange chromatography so in this method the beads in the column are going to be charged are going to be coded with charged substances so they will attract or bind compounds that have opposite charge so for instance a positively charged so if you have a column all right and you have these beads all right and you have a a positively charged column bead right column beads they're going to attract and try to hold on to negative proteins all right as it passes through the column so that's one way of holding so so what this means is if you have a positively charged coded substance right if you if the beads in the column are coated with a positive charge positively charged substance and it's going to bind to your to negatively charged proteins that means your positive proteins are going to move much faster and they're going to be eluded first they're going to be they're going to come out first from your column and then in comparison to those negatively charged proteins which are being bound to your positively charged coded column so that's one thing to keep in mind then you have size exclusion chromatography in this method the beads that are used in the column are going to contain tiny pores of varying sizes and these tiny pores are going to allow small compounds to enter the beads and that's going to slow them down so the larger compounds that can't fit into the pores they're going to move and travel through the column faster than the smaller compounds that will fit into the pores of the beads and and kind of stay there for longer so those three types of chromatography that we've talked about column chromatography ion exchange and size exclusion one more thing in the realm of protein analysis and protein separation that we want to talk about is X-ray crystallography and NMR so separating from separating proteins from one another is generally only the first step in analysis the next step is to then study the isolated protein protein function structure or quantity is often of high interest to researchers right especially if they're studying a specific protein that they want to learn everything about even after protein separation and even after protein identification protein analysis tools can be used to study that protein further now protein structure specifically it can be determined through things like x-ray x-ray crystallography and also NMR nuclear magnetic resonance spectroscopy now before crystallographic analysis though that protein has to be isolated and it has to be crystallized x-ray crystallography though is the most reliable and common method for that 75 of the protein structures that are known today were actually analyzed through this method and essentially crystallography measures electron density on an extremely high resolution scale it can also be used in addition on nucleic acids and x-ray diffraction pattern is generated using this method all right something that's going to look like this some diffraction pattern the small dots in the diffraction pattern can then be interpreted to determine the protein structure you don't necessarily need to know how this exactly works because it's very complicated but you should know that x-ray crystallography is one of the most commonly used methods to determine protein structure and what you get from x-ray crystallography is an x-ray diffraction pattern that allows you to determine the protein structure from you don't need to know how that works that is complicated just that it does work all right and with that we end our review of this chapter next video we'll do some practice problems and that's it you're prepared for the MCAT biochemistry section let me know if you have any questions comments or concerns down below other than that good luck happy studying and have a beautiful beautiful day future doctors