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Module 1: Proteomics

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Amino Acids and Proteins

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Welcome to MOOC NPTEL course on bioengineering, an interface with biology and medicine.Last week, we studied few basic concepts for cell cycle and development, we also saw manyinteresting examples, this week we are going to discuss about amino acids, proteins andprotein related technology especially, how they have driven the field of proteomics.So, let us start with some very basic fundamental concepts for amino acids and proteins.And then, we will slowly build these concepts further and then see how they can be utilizedfor various new tools and technologies to study proteins in a very high throughput manner.So, first of all, why studying proteins are crucial, proteins perform a range of cellularfunctions and as a result they are the one which most accurately reflect what is goingon inside the human body or physiology, disease results as a part of protein malfunction,so most of the drugs currently either are depending on the protein function or theirprotein themselves, so therefore studying proteins becomes very crucial.Again to remind you from the central dogma which we talked in the beginning which reflectsthat you know starting from DNA to RNA, the process of transcription governs that andthen from RNA to protein, it is a translation process and then again protein gets furthermodified in the post translation modification and, and that kind of you know, gives sortof functionality to the proteins.So, what are amino acids; amino acids are building block of proteins, the basic monomericunit of polypeptides and proteins, there are 20 standard amino acids which are having uniquestructures and properties that can be combined in multiple ways to make up the wide rangeof proteins known to us, each amino acid is specified by 3 letter and single letter code.The amino acids they are linked to each other with peptide bonds.And they form eventually, the polypeptide proteins, here we have shown a basic aminoacid structure with a minor group, carboxyl group and R side chain which keeps changingin different amino acids.Let us briefly start with the broad category of amino acids, starting with aliphatic aminoacids, these are large and diverts hydrocarbon side chains which enables them to form thecompact structures.Some examples of these aliphatic amino acids include valine, leucine, isoleucine methionineand prolene, these are large aliphatic side chains and hydrophobic, the water solubleproteins are stabilized by the hydrophobic effects and then therefore, they have a tendencyto form the clusters because of the hydrophobic groups.Here, I have shown you the structures of these amino acids, which are nonpolar side chains,hydrophobic amino acids, starting from the glycine, the simplest one which is Achiralamino acid and you can see only hydrogenase in the side chain R group and then Alanineis having methyl group CH3, then we have valine when we have CH3 CH3 and CH group then, wehave further methyl groups being added in leucine and the isoform of that which is isoleucine.Then, we have methionine which is a cell for containing amino acid includes thioether group,then we have phenyl alanine with the phenyl group, tryptophan rings which is characteristicof tryptophan amino acid and then, we have proline, which is not having any free aminogroup and they have a ring structure which provides some sort of conformational restrictionand that gives some sort of uniqueness to a amino acid protein.Now, let us look at the polar side chain based amino acids which are hydrophilic in naturefor example, serine; serine is having a CH2OH group, it resembles like Alanine but havingunique a hydroxyl group; OH group, then we have Throenine which resembles with valinebut it also has the hydroxyl group, so it an additional asymmetric center, then we havea cysteine amino acid which is having sulfhydryl or thiol groups.Then tyrosine amino acid, asparagine and glutamine which has these CH2 CNH2O bond, then comingto electrically charged side chain or hydrophilic amino acids, we have Aspartic acid and glutamicacid, which are acidic and having negatively charged groups, then we have a lysine arginineand histidine which are having the positive charge and these are basic amino acids, theyare again characterized with in the case of lysine with amino group.In case of arginine, with the guanidinium group and case of histidine having one imidazolegroup, so I am going to talk about some of these amino acid structures in some more detailin the animation.But for the time being let us you know, focus on aromatic amino acids looking at these 3;phenylalanine, tyrosine and tryptophan, the phenylalanine has a phenyl ring as you cansee in the structure, tyrosine having a reactive hydroxyl group and then tryptophan is havingindole ring which are you know, 2 fused rings are there with NH.So, now these kinds of properties of these aromatic amino acids, they give them the hydrophobiccharacteristics like especially in case of the phenylalanine.Whereas, the tyrosine and tryptophan they are more hydrophilic because of the presenceof OH and NH group, these aromatic amino acids are also being utilized for you know, measuringthe concentrations in the protein and people look into the absorption spectra of tyrosineand tryptophan.So, I shown here on the slide, the tryptophan and tyrosine aromatic rings they stronglyabsorb at the UV light and the light absorption is a characteristic which can be used forthe protein estimation.So, as I mentioned that you know these amino acids they come together to, to form peptidebonds and the peptide bond is formed during the process of linking together these aminoacids with the carboxyl group of one amino acid being linked to the amino group of anotheramino acid with the loss of a water molecule.Let us now review some of these concepts in an animation.Amino acids are the building blocks or monomers that make up proteins; they consist of a centralalpha carbon atom bonded covalently to an amino group, a carboxyl group, a hydrogenatom and a variable side chain also called the R group.Amino acids are the basic monomeric constituents of proteins found in varying amounts dependingupon the type of protein; they are classified based on the properties of their side chainsor R groups which vary in size, structure and charge.The polarity of the side chains is one of the main bases for classification.Amino acids having nonpolar aliphatic side chains include glycine.Alanine.Proline.Valine.Leucine.Isoleucine.And methionine.Essential amino acids are those that cannot be synthesized de novo in the organism andtherefore, must be included in the diet.Non-essential amino acids on the other hand can be synthesized from various precursors.Serine.Threonine.Aspargine.Glutamine.And cysteine consists of polar but uncharged side chains.Lysine.Arginine.And histidine have positively charged side chains.Aspartic acid.And glutamic acid are polar and negatively charged amino acids.Tryptophan.Tyrosine.And phenylalanine are all essential amino acids having an aromatic side chain.Amino acids are the building blocks or monomers that make up proteins, amino acids are orientedin a head to tail fashion and linked together such that the carboxyl group of one aminoacid combines with the amino group of another, 2 amino acids joined together by means ofsuch a condensation reaction with a loss of a water molecule forms a dipeptide.Many such amino acids linked together form a polypeptide.The peptide bond is rigid due to its partial double bond character arising from resonancestructures however, the bonds between the alpha carbon and amino and carboxyl groupsare pure single bonds that are free to rotate.What are the amino acid properties in relation to isomerism, so let us talk about opticalisomerism, just imagine the chiral molecules they interact with the plane polarised lightin such a manner that they can rotate the plane of polarization either in the clockwiseor the counter clockwise directions.So, depending on that in which direction the molecule rotates, the plane of the polarizationcan be designated as plus, you can say dextrorotatory or you can say levorotatory, this nomen clatureis not exactly same as the D and L designations, which actually refers to the absolute configurationsis specified on the basis of their relationship with D and L glyceraldehyde.Majority of the amino acids which are found in the proteins are of the L configurations.Let us review these concepts in the following animation.Before learning about the isomerism, let us first know what chirality is; the term chiralityarises from the Greek term care meaning, handedness, just like the 2 hands are non-superimposablemirror images of each other, amino acid molecules are also non superimposable due to their chiralalpha carbon centre.All amino acids except glycine contain an asymmetric centre that makes them chiral innature due to which they can rotate the plane of polarized light.The 2 enantiomers designated as D and L rotates the plane of polarization in opposite direction.The 2 enantiomers of amino acids are non-superimposable mirror image due to the spatial arrangementof 4 different groups about the chiral carbon atom, rotation of either isomer about itscentral axis will never give rise to the other isomeric ring structure.What are the acid and base properties of amino acids?All amino acids they exist in the completely protonated forms in the acidic medium, whichis known as the cationic form.Both amino and the carboxyl groups are protonated and this state in which the amino acids hasno net charge that is known as the deuteron, it is neutral because of the presence of NH3+and COO- groups.What is the anionic form of amino acid?In a highly alkaline medium, all amino acids exist in the anionic form because of the presenceof COO- groups, so looking at these properties of cationic and anionic forms and deuteronswhich are formed one could do titration curve.So, the number of equivalents of alkali being consumed during the process of addition ofalkali to the amino acid solution is plotted against pH of the solution in the flask whichis the unique titration curve of each amino acid.The titration curve depicted corresponds to that of glycine.Let us review this concept in animation.Amino acids in acidic medium exist in the completely protonated form carrying a netpositive charge.This can be confirmed by means of simple paper electrophoresis, the sample solution is appliedat the center of the strip.And current is passed through it.The colourless amino acid solution can be detected by spraying the strip with ninhydrinwhich gives it a purple colour; migration of the spot towards the negatively chargedcathode confirms the net positive charge of the amino acid.All amino acids exhibit a characteristic titration curve with distinct PK values, 0.1n NaOH isadded to the acidic amino acid solution.The cationic form of the amino acid is gradually converted into its neutral.Or zwitter ionic form by loss of a proton from its COOH group, this can again be confirmedby electrophoresis where there is no migration of the samples spot.Number of equivalence of alkali being consumed is plotted against the pH of the amino acidsolution to obtain the titration curve, pK1 of glycine is found to be 2.34 that is, itstarts to lose its carboxyl group proton at this pH.Removal of the proton from the amino group constitutes the second stage of the titrationcurve; continued addition of alkali to the amino acid solution gradually converts thezwitter ionic form into the anionic form.Migration of the sample spot towards the anode during electrophoresis confirms this.The pK2 of an amino acid is obtained by continued addition of alkali to the neutral solutionof the amino acid, pK2 of glycine is found to be 9.6, some amino acids having positivelyor negatively charged side chains will have pK1, pK2 and pKr, which corresponds to ionizationof the side chain, these amino acids have good buffering capacity around 1 pH unit oneither side of the pK values.After discussing about amino acids, now let us move on to the proteins, so what are proteins?These are linear polymers which are built of monomers of amino acids and they have widerange of functional group which accounts for various protein function and then that reactiveproperties are very crucial for the enzyme and the protein function.There are many properties like protein-protein interaction, protein bio molecular interactionwhich generates a synergistic capability which may not be obtained just by studying an individualprotein.And therefore, it becomes very crucial to study proteins together and then, you knowone need to understand the in totality, proteins are governing that function.So, understanding protein function is key to biology, let us look at some of these keycharacteristics here like enzyme catalysis, where enzymes catalyses all the biochemicalreactions by increasing rate of reactions, so enzymatic catalysis is the process whenenzymes catalyse all biochemical reactions by increasing the rate of reactions.There are proteins which are involved in the transport and storage processes.The proteins which can transport the small molecules like oxygen, iron etc. proteinsare also involved in doing the coordinated motion for example, muscle contraction, bacterialchemotaxis, chromosomal movements, sperm propulsion etc. all of these are coordinated motion whichare governed by different proteins, where its mechanical strength is also governed bythe different proteins.For example, with skin and bones by the collagen proteins and here, with the keratin protein,I discussing about protein function, proteins are also involved in the immunity processfor example, antibodies which exemplify the specificity of protein, protein and proteinligand interactions.They are also involved in neurotransmission which is in the response of cells to thesestimuli in the nerve cells.In the process of growth and differentiation for example, the transcription factors; theyare involved in the gene expression processes during growth and development.So, I am sure you appreciate that you know, proteins play key structural and functionalrole and they define you know, wide range of functions involved in signalling, transport,catalysis, movement, structure, regulation etc.Let us now discuss about different levels of protein structures.So, the primary structure refers to the sequence of amino acids, the secondary structure refersto the locally folded regions.The tertiary structure refers to the overall folding of the protein structures and quaternarystructure refers to all the interaction between individual protein subunits in a multi subunitcomplex.Let us start with each of these structural details of proteins, let start with primarystructure.The sequence of amino acids are joined together by the peptide bond, which forms a linearpolymer constituting the primary structure of the protein, the linear polypeptide chainsare often cross-linked, most commonly by the cysteine bonds and then they are linked togetherto form a cysteine unit.The first primary structure that was deduced that was for a protein insulin, which wasdiscovered by scientist Frederick Sanger.So, what does the significance of these, you know primary structure or the amino acid sequences.It is essential for the elucidation of its mechanism of action, it also determines the3 dimensional structure of the proteins, the amino acid alteration can produce certainabnormalities in the individuals and diseases like you know, sickle cell anaemia is oneof the example in which how the amino acid alteration could you know, just for singleamino acid change could lead to you know, certain abnormality.The sequences also tell us an evolutionary history of the protein and lot of evolutionaryrelationship of organisms could be established looking at their amino acid sequences.Let us now talk about secondary structures; the folding of a polypeptide backbone by meansof internal hydrogen bonds between nearby amino acid residues that gives rise to a regulararrangement which is defined as the secondary structure of proteins.The different type of alpha helices and beta sheets which are most commonly observed inthe secondary structure of proteins due to their highly favourable phi and psi angleswhich is described by the Ramachandran plots.The amino acid prolene, it tends to disrupt the helix and it is often found you know,bending in the structure which is known as the reverse turn or the beta bends, let uslook at the alpha helices first.So, proteins have variable helices contents.The alpha helices is a rod like structure, the main chain is tightly coiled around helicalaxis and the side chains they are extended outwards which is away from the helical axisthat specific hydrogen bonds which can stabilize this helical codes to make alpha helices bonds.The alpha helix is formed in the regions which are stabilized by the hydrogen bonds betweenatoms of the polypeptide backbones and residue the carboxyl group of the, the first aminoacid with the NH group of the fourth is forming this bond and similar thing of the alpha helixcould be seen in different examples whether it is a keratin protein or it is collagenprotein, when you can see even double helix or triple helix is being present and theyactually you know provide a lot of a strength to these protein structures.Beta sheets; these are another common periodic structural motives, which are fully extendedstructure, they are parallel or antiparallel and we can see the structures of startingfrom you know the parallel and the antiparallel beta strands, so the beta pleated sheet iscomposed of 2 or more polypeptide chains shown in this on the screen is the beta strand onthe top and the beta sheet.One of the examples shown here is the spiders silk fiber which is one of the structuralprotein which contains beta pleated sheets, let us move on to tertiary structures now.The tertiary structures refers to the interactions especially hydrophobic, electrostatic, hydrogenbonds etc. between amino acid side chains which are located far apart in the polypeptidesequence and that causes the protein to fold resulting into a 3 dimensional arrangementof atoms which is known as tertiary structure.The folding takes place in such a manner that the hydrophobic residues they get buried toform the core while the hydrophilic amino acids they remain on the surface in contactwith the polar surroundings, so there are numerous interactions which specialises atertiary structure of the proteins.And that is shown here in some of the examples especially for the myoglobin and transthyretinproteins, these structures could be studied using NMR and x-ray crystallography whichprovides the detailed 3 dimensional structures.So, what are quaternary structures?Many proteins have more than 1 polypeptide chains also called a subunit that are assembledtogether by various interactions like electrostatic, van der Waals, disulphide bonds and that givesrise to the quaternary structure.So, quaternary structure is referring to the interactions between individual protein subunitsin a multi subunit complex and it actually, provides a final level of protein structure,it also depicts the special arrangements of subunits and their interactions and how thesepolypeptide chains assemble to form these multi sub unit structures.Shown here are the examples of hemoglobin and transthyretin.So, this is how we can summarize these different levels of protein structures, we have primarystructure, secondary structure, tertiary structure and the quaternary structure.Let me explain you this in more detail in the following animation.Amino acids are joined together in a head to tail arrangement by means of peptide bondswith a release of water molecules; this linear sequence of amino acids constitutes the primarystructure.The folding of the primary structure into the secondary is governed by the permissiblerotations about the Phi and Psi angles, not all values of these angles lead to stericallyfavourable confirmations.The Ramachandran’s plot defines these regions of favourability.Amino acids along the polypeptide backbone interact through hydrogen bonds leading tosecondary structures, the alpha helix has intrachain hydrogen bonds between the H ofNH and O of CO in every fourth residue.Most alpha helices are right handed since this conformation is energetically more favourable.The amino acid proline which has a cyclic side chain, does not fit into the regularalpha helix structure and thereby, limits flexibility of the backbone, it is commonlyreferred to as the helix breaker.[Amino acids along the polypeptide backbone interact through hydrogen bonds leading tosecondary structures; the alpha helix has intra chain hydrogen bonds between the H ofNH and O of CO in every fourth residue.Most alpha helices are right handed since this conformation is energetically more favourable,the amino acid proline which has a cyclic side chain does not fit into the regular alphahelix structure and thereby limits flexibility of the backbone, it is commonly referred toas the helix breaker.]Alpha helices can also wind around each other to form stable structures such that theirhydrophobic residues are buried inside while the polar side chains are exposed to the aqueousenvironment.Alpha carotene, the major protein component of hair consists of 2 such coiled coils forminga left handed super helix, collagen which is a fibrous component of skin, muscle etc.consists of 3 such coiled alpha helices.It has a characteristic recurring amino acid sequence of glycine, proline, hydroxy prolinewith glycine appearing at every third residue.Beta pleated sheets discovered by Pauling and Corey is another common secondary structurewith periodic repeating units, it is composed of 2 or more polypeptide chains with theirside chains oriented above and below the plane, it is an extended structure with hydrogenbonds between the chains stabilizing it.Amino acids in parallel beta sheets which run in the same direction interact with 2different amino acids on the adjacent strand through hydrogen bonds; amino acids in anti-parallelstrands on the other hand interact with only one amino acid on an adjacent strand.Almost all proteins exhibit a compact globular structure which is possible only if thereare turns or loops between the various regions, beta turns, which are the most commonly observedturned structures consist of rigid well defined structures that usually lie on the surfaceof the protein molecule and interact with other molecules.A combination of secondary structures such as the helix turn helix which consists of2 alpha helices separated by a turn is also observed and these are known as super secondarystructures or motives.Amino acids located far apart on the polypeptide chain interact with each other by means ofhydrogen bonds, electrostatic interactions, disulphide bridges etc. allowing the proteinto fold 3 dimensionally in space giving rise to the tertiary structure.Folding takes place such that the hydrophobic residues are buried inside the structure whilethe polar residues remain in contact with the surroundings.The tertiary structure of myoglobin determined by John Kendrew clearly revealed that thenature of amino acid side chains dictate their location in the tertiary structure, hydrophobicresidues are found buried inside the structure, while the polar amino acids are found on thesurface, 70% of the main chain of myoglobin is folded into alpha helices with the restbeing present in the form of turns and loops which are essential to give it a compact structure.Different subunits or polypeptide chains interact with one another and are held together bymeans of ionic, electrostatic van der Waals etc. interactions, such multi subunit proteinsare said to have a quaternary structure, the final level of protein structure.So, today in this lecture I try to refresh you about some of the basic concepts of aminoacids and proteins of course, this whole subject needs not more detailed study.But you know just to give you some basics and some idea, the kind of diverts side chaingroups present in amino acids and why they are so crucial and why these studying proteinsare so difficult because of the so much uniqueness in these amino acids and 20 different forms,they give rise to many different type of proteins and studying the proteins they therefore,becomes on one hand very crucial but also becomes very challenging.In case of DNA studies, we had only 4 base pairs to study ATGC, in case of amino acidswe have 20 combinations to a study, we do not have the techniques like polymerase chainreaction which we have studied earlier, in case of DNA technologies which could justsimply amplify the DNA molecule, we do not have techniques like that in the case of theprotein technologies maybe you can start with you know small amount of protein and amplifythat to obtain the huge amount of protein.So, these are many challenges to study the proteins and proteins are usually very dynamic,they are very labile, sometime they are you know, very short life, so studying proteinsbecomes very challenging and therefore, knowing their basics and properties are very crucial.I just try to give you some glimpse of these structure of the different levels of proteinstructures; primary, secondary, tertiary and quaternary and intention is that after reviewingsome of these basic concepts, you should go back, read the textbooks in much more detailand then you are more prepared to actually learn the tools and techniques which are employedto study proteins especially for the various type of protein research and proteomics research.So, in the next couple of lectures, I am going to talk about different technologies, howthey are trying to study the proteins and why, you know the complex protein mixturesespecially at the proteome level and to do that different type of protein propertiesare being utilized.So, in the next couple of lectures we have more you know technologybased understanding and then these basic concepts will be very useful over there, thank you.