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Fundamentals of Biochem

by: Breyannah Sally

Fundamentals of Biochem Cbio 390

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polypeptides and protiens
Intro to Biochemistry
Dr. Musey
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This 61 page Bundle was uploaded by Breyannah Sally on Friday September 9, 2016. The Bundle belongs to Cbio 390 at Clark Atlanta University taught by Dr. Musey in Fall 2016. Since its upload, it has received 6 views. For similar materials see Intro to Biochemistry in Chemistry at Clark Atlanta University.


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Date Created: 09/09/16
Amino Acids, Peptides & Proteins Modules 06740 / 06741 / 06742 1 Course Content 1. Introduction to amino acids and peptides 2. Separation and purification of amino acids and proteins 3. Chemical and enzymatic reactions of amino acids and peptides 4. Polypeptide sequencing 5. Peptide synthesis 6. Protein structure General Books Most general “Organic Chemistry” text books have chapters covering many aspects of the course. Similar biochemistry texts have similar sections / chapters. Specialist texts (include): “An introduction to peptide chemistry” by P.D. Bailey “Amino acid and peptide synthesis” by J. Jones. 2 1 Peptides and Proteins monomers dimers to oligomers polymers amino acids (poly)peptides proteins function function function the building blocks hormones enzymes (catalysis) neurotransmitters receptors (binding) (etc) switching structural 3 Peptides and Proteins amino acids (poly)peptides proteins MW = 75~204 g/mol 2 to ~50 amino acids ~50 amino acids+ (for common examples) 2 to ~10 = peptide 11 to ~50 polypeptide polypeptides may Proteins almost always have longer range have well defined structure or order structures with higher order (2 , 3 and 4 structure) The MW of proteins are often expressed in Daltons (D) or kiloDaltons (kD) 4 2 Primary Structure ▯ The sequence of amino acids that makes up the chain of a peptide or protein is called the primary structure of that peptide. AA -AA -AA -AA -AA -AA -AA …. 7 ▯ There are 20 amino acids encoded in our DNA, but many more less common ones. 6 ▯ … 20 (= 64 million) possible hexapeptides could be made from these 20 amino acids. 5 Amino Acids NH 2 e d R H H2N g b b a H 2 CO 2 a CO2H H2N a CO2H a-amino acid b-amino acid a,e-diamino acid R is known as “the side chain”, which often contains a functional group. ‘w’ is often used to denote a functional group at the end of a (side) chain (of unspecified length) There are twenty common variants for R “encoded” in our DNA (22 actually) all of which are a-amino acids 6 3 DNA-encoded Amino Acids A R N D C E Q G H I Ala Arg Asn Asp Cys Glu Gln Gly His Ile CH CH CH CH3 CH2 CH2 CH2 CH2 CH2 CH2 H 2 3 CH CH2 C O C O SH CH2 CH2 2 N CH R H CH2 NH2 OH C O C O NH 3 H2N CO 2 NH OH NH2 C NH NH2 L K M F P S T W Y V H Leu Lys Met Phe Pro Ser Thr Trp Tyr Val N CO 2 H CH2 CH2 CH2 CH2 CH2 CH2 CH OH CH2 CH 2 CH CH3 CH CH3 CH2 CH2 CH2 OH CH3 CH3 N CH3 CH2 S CH2 NH CH2 CH3 NH2 OH 7 Some amino acids and dipeptides ▯ g-Amino butyric acid H2N CO 2 ▯ GABA ▯ neurotransmitter NH 2 ▯ Monosodium glutamate ▯ “Meaty” flavouring additive (umami) HO C CO 2a 2 ▯ Also a neurotransmitter! CH CO H 2 2 O ▯ Aspartame H ▯ Artificial sweetener N H N OMe ▯ Simple dipeptide based on aspartic 2 acid and phenylalanine O CH Ph 2 ▯ Penicillin G Ph H N ▯ Antibiotic N S ▯ Dipeptide-based with further modification O N Cysteine O Valine CO H 8 CO 2 4 Peptides ▯ Enkephalins are a family of peptides ▯ They are present in the brain and are involved in the control of pain sensation. OH peptide ▯ E.g. (amide) bond residue O O H H N N OH H2N N N N terminus H H C terminus O residue O O ▯ Convention dictates that the amino (N) terminus is always drawn on the left and the carboxy (C) terminus on the right. 9 Conventions Short hand notation is used to describe peptide and polypeptide / protein primary structure. CO H 2 O H ▯ With short peptides and amino acids H N N OH the three- letter code often used. 2 Defined sequences are separated by O Ph either a dot or a dash. Asp-Phe or Asp.Phe ▯ As there is a strict convention that oligo- and polypeptides are always OH N C written with their N-terminus on the left and C-terminus on the right Asp.Phe, for example, is not the O O same as Phe.Asp N N OH H2N H H O O O ▯ For polypeptides and proteins the Tyr.Gly.Gly.Phe.Leu one-letter code is most often used, without any dots or dashes. or YGGFL 10 5 Conventions O NHAc = Ac We use an extended notation to H3C indicate if the termini are modified. CO H ▯ Aspartame, a methyl ester, 2 would be H-Asp.Phe-OMe Ac N CO H H N CO Et H 2 2 2 ▯ N-acetyl aspartic acid would be: Ac-Asp-OH H-Lys(Ac)-OEt ▯ Side-chain functionalisation is denoted in brackets. CO2H Ph H O H O Amino acid abbreviations are separated N N by commas and given in brackets H2N OH H2N OH O O indicate the peptide composition, not Ph CO2H necessarily the sequence. Asp.Phe Phe.Asp ▯ Thus (Asp,Phe) could refer to either dipeptide Phe.Asp or Asp.Phe (Asp,Phe) 11 Most amino acids are chiral Many amino acids possess (at least) R H R H one chiral centre. H 2 CO 2 HO 2 NH 2 ▯ All 20 DNA-encoded a-amino acids are laevorotatory (L), except glycine laevorotatory (L) dextrotatory (D) (S) (R) which is achiral (R = H). C.I.P. rules reveal that all such (L) amino acids are (S) except cysteine which is (R). Here the D-amino acids are also side chain is CH2SH and S takes precedence over CO H. very common in nature 2 L-Cysteine R H N H S sugar-peptide- (D)-Ala-(D-Ala O N penicillins O D-Valine in bacterial peptidoglycan CO2H 12 6 Acid-base properties Amino acids possess both acidic and basic functional groups K H H RCO 2 1 RCO 2 H pKa 1 2.2 H 2 CO 2 at high pH a negatively charged carboxylate anion is obtained K2 pKa ~ 9.0 pH ~ 6-7 RNH 2 H RNH 3 2 at low pH a positively charged ammonium cation is obtained R H at about neutral pHs a zwitterion exists H3N CO 2 (German: zwitter = hybrid) The pH at which the amino acid‘s net charge is zero is called the isoelectric point (IEP, sometimes IP) 13 Isoelectric Point pH ▯ The acid-base titration curve for glycine (IEP=6.0) shows two ionisations: one for the pKa 2 acid and one for the amine. 9.0 ▯ The isoelectric point will vary depending on the inductive 6.0 5.5 IEP effect of the side chain R. pKa 1 ▯ In phenylalanine (IEP=5.5) the 2.0 -I side chain will lower the pKa of both acid and amine group very slightly compared to glycine (R=H) 0.5 1.0 1.5 equivalents of NaOH added 14 7 Amino acid solubility Due to their zwitterionic nature amino acids are more readily soluble in polar solvents than non polar solvents, but side chain groups will affect relative solubility. Amino acid Serine Glycine Phenylalanine Side chain R = CH OH 2 H CH 2h polar non polar Solvent H 2 Ser > Gly > Phe CHCl 3 Ser ~ Gly ~ Phe Benzene Ser < Gly < Phe 15 Amino acids grouped by side chain A R N D C E Q G H I Ala Arg Asn Asp Cys Glu Gln Gly His Ile CH CH 2 CH CH3 CH3 CH2 CH2 CH2 CH 2 2 CH 2 H CH C O C O SH CH2 CH CH2 2 2 N CH2 NH2 OH C O C O CH3 POLAR NH NH OH NH 2 ACIDIC C NH BASIC NON-POLAR NH2 L K M F P S T W Y V Leu Lys Met Phe Pro Ser Thr Trp Tyr Val O CH2 CH2 CH2 CH 2 C OH CH 2 CH OH CH2 CH 2 CH CH3 CH CH CH3CH2 CH2 OH CH3 3 HN CH3 CH2 S NH CH CH 2 3 NH2 OH 16 8 Basic and Acidic Side Chain Functional Groups O C ▯ Aspartic and glutamic acid CO2 2 have acid side chain functional H H groups (carboxylic acids). H N CO ▯ Aspartic acid IEP = 2.98 3 2 H N CO 3 2 ▯ Glutamic acid IEP = 3.22 Overall - 1 charge at pH 7 ▯ Lysine and arginine have basic NH2 side groups (amine and H3N H 2 guanidine respectively). NH ▯ Lysine IEP = 9.76 ▯ Arginine IEP =10.76 H ▯ The imidazole ring found in H histidine is only weakly basic (IEP = 7.59) H3N CO2 H3N CO 2 Overall +1 charge at pH 7 17 Titration curves for lysine and glutamic acid pH ▯ The acid-base titration curve IEP pKa for lysine shows three 3 10.5 ionisations: one for the acid pKa and two for the amines. 2 9.0 pKa 3 ▯ The acid-base titration curve for glutamic acid shows three ionisations: two for the acids 4.5 and one for the amine. pKa 2 2.0 pKa 1 ▯ The isoelectric point will pKa IEP correspondingly be high for 1 lysine and low for glutamic acid. 0.5 1.0 1.5 2.0 2.5 3.0 equivalents of NaOH added 18 9 Separation and Purification of Amino Acids & Proteins 19 Paper Chromatography - analysis ▯ Like conventional TLC using silica coated plates, the cellulose in paper can be used as a stationary phase for the chromatography of polar molecules such as amino acids. ▯ Eluants, the composition of which is crucial to separation, often contain: ▯ Water polar solvent which binds strongly to the polysaccharide in cellulose ▯ Butanol solvent miscible with water but which decreases the overall dielectric constant ▯ aq. Ammonia to increase pH ▯ Acetic acid to decrease pH ▯ 2-Dimensionsional chromatography is often used for tricky separations by utilising different eluants for each direction. 20 10 Paper chromatograms first elution 2D chromatogram eluting with a different solvent mixture in each direction second elution 21 Ninhydrin – detection of amino acids ▯ Amino acids can be detected on the chromatogram by using ninhydrin. A solution of ninhydrin is sprayed onto the paper and heated. The amino acids show up as purple spots (proline appears yellow). R O O O R OH H2O H 2 CO2H O O N H OH O O ninhydrin O O -C2 / H+ OH O OH O R ninhydrin H2O / H+ N NH2 N O O O O lmax70nm 22 11 Gas Chromatography – analysis ▯ Amino acids can be analysed by standard GC methods, but due to their zwitterionic nature they must be derivatized to increase their volatility. ▯ Typical derivatization includes esterification of acid groups and trifluoroacetylation of amines (on side chains too if necessary). Silylation can be used for both groups. O R R OEt Me3Si OSiM3 F3C N N H H O O ▯ An increasing temperature gradient is used to make sure elution times are reasonable. ▯ GC machines can be calibrated to give quantification and linked to a mass spectrometer to help in identification. 23 Separation of proteins: dialysis ▯ Proteins can be separated using dialysis. Using semi-permeable membranes, which only allow molecules up to a certain size to pass through the them, complete partition of proteins with a similar MWs is not possible. ▯ As the overall range of protein MWs is large this technique is a very useful, if crude, method to separate higher and lower MW materials as a first stage in purifi.ation 24 12 Ion-Exchange Chromatography – preparative separation As amino acids have a net charge which varies with the pH, this property can be used to separate amino acids using ion-exchange resins X Me3N SO3Na functionalized polymer bead columns packed with polymer (resin) beads Anion exchange resin Cation exchange resin Anchored group is +ve Anchored group is -ve 25 Ion-Exchange Chromatography ▯ Peptides and amino acids with net negative charges bind to anion exchange resins, but those with positive charges are repelled by the resin and are eluted quickly. ▯ The reverse is found for cation exchange resins ▯ Amino acids and peptides have different net charges at different pHs. ▯ Side chain functional groups are a big factor in determining the net charge and therefore the IEP; this is true for peptides and proteins as well as their individual amino acids constituents. ▯ The pH of the eluant can be changed with time. This will change the overall net charge of the peptide and therefore its “stickiness” to the resin. 26 13 Separation of proteins: gel filtration or size exclusion chromatography Proteins come in a wide variety of shapes and sizes. Gel filtration chromatography uses porous resins to separate proteins on the basis of these differences. It is sometimes know as size exclusion chromatography. Residency times in the pores of the resin is greatest for the smallest proteins. Larger proteins elute more Column with quickly. macroporous resin beads 27 Gel filtration chromatography - problems and solutions ▯ If the resin has pores of one size only, then separation of a complex mixture of proteins will be inefficient. ▯ ……either the proteins enter the pores or they do not, resulting in two elution peaks only. ▯ If a complex mixture or proteins are to be separated then the macroporous resin will need to have a range of pore sizes. ▯ ….but if the range of pore size is too great then the resolution is lost and the elution bands are broadened resulting in overlap. ▯ The compromise is to have a narrower range of pore sizes, approximating to the size of the protein of interest. Like with dialysis other techniques are needed to isolate the peptide in a really pure state. 28 14 Affinity chromatography Affinity chromatography uses porous resins beads covalently attached to which is the natural ligand of the protein of interest. Elution of the protein mixture results in binding and retention of the single protein whilst the others elute normally. Add to eluant here Natural ligand ( ) Addition of excess, covalently bound to resin unbound natural ligand to bead traps complementary the eluant displaces Protein protein as it passes over theprotein from resin and mixture bead. allows elution. Affinity column 29 Electrophoresis ▯ Electrophoresis is a technique that uses the net charge of peptides (amino acids) as a basis for separation. ▯ A potential difference is applied across a solid material (e.g. paper for amino acid analysis) permeated by an electrolyte. Lys.Lys Phe.Lys Phe.Asp Asp.Asp Support mixture material applied here impregnated with electrolyte Cathode Anode cations anions ▯ Anions migrate to the anode and cations to the cathode. The rate of diffusion is related to the size and net charge. Small highly charged proteins migrate more quickly. 30 15 Isoelectric focusing (IEF) ▯ It is difficult to separate two peptides or proteins of similar MW if they differ only slightly in their net charge at a given pH. ▯ This problem can be overcome by performing the electrophoresis across a pH gradient – known as isoelectric focusing. ▯ As soon as the IEP is reached the proteins carry zero net charge and so stop migrating. 31 Electrophoresis equipment typical IEF equipment electrodes electrophoresis ‘support’ material ceramic cooling plate 32 16 SDS-PAGE NH PAGE is polyacrylamide gel electrophoresis 2 acrylamide SDS is sodium docdecyl sulphate - a surfactant O SDS SO 3a ▯ The support material for SDS-PAG electrophoresis is a cross-linked gel formed by the polymerisation of acrylamide and a cross-linking agent. ▯ Adding the surfactant SDS causes proteins to unfold (denature). ▯ The surface of the protein is “coated” with negatively charged SDS molecules in (approx. one SDS per two amino acid residues). ▯ All proteins are given an overall negative charge and so migrate to the anode. ▯ Adding the surfactant means that proteins migrate as a function of their MW. Larger proteins migrate more slowly due to their bulk (despite their high net negative charge) whereas small proteins migrate more quickly. 33 2-Dimensional gel electrophoresis ▯ Proteomics is the name given to the recent scientific discipline which is based around the study of the entire set of proteins produced by a cell (known as the proteome, c.f. genetics and genome). ▯ The aim is to study the overall effect on protein expression in a cell by, for example, the action of a drug or other bioactive compound. ▯ 2D-electrophoresis (c.f. 2D amino acid chromatography) is needed in an attempt to separate the many hundreds or perhaps even thousands of proteins that may be present in a cell. ▯ The two techniques most commonly used are I.E.F. followed by SDS- PAG electrophoresis. ▯ Analysis of proteins is performed using MALDI - a new(-ish) mass spectrometry ionisation technique which uses lasers (Matrix Assisted Laser Desorption Ionization). 34 17 Example of a 2D gel IEF SDS 35 Chemical and Enzymatic Reactions of Amino Acids & Peptides 36 Acid and alkaline hydrolysis ▯ Peptides can be cleaved into their constituent amino acids by acid or alkaline hydrolysis of the peptide (amide) bond. ▯ This reaction follows the same standard mechanism of hydrolysis of a simple amide bond using either acid or alkali. ▯ Acid conditions: 6M HCl, 110 C, 24 h, sealed and evacuated tube. ▯ Methionine (Met) and cysteine (Cys) are easily oxidised. The reaction tube must therefore be degassed and flushed with nitrogen (to remove any dissolved oxygen) before evacuation and sealing. ▯ Tryptophan (Trp) is totally destroyed under these conditions. 37 Acid and alkaline hydrolysis ▯ Acid: (cont.) ▯ Serine (Ser) and threonine (Thr) are slowly dehydrated to alkenes but losses are not usually serious. ▯ The side-chain amides of glutamine (Gln) and asparagine (Asn) are hydrolysed. Analysis of constituent amino acids will therefore indicate higher levels of glutamic (Glu) and aspartic (Asp) acids. ▯ Alkali conditions: 2M NaOH, 100 C. ▯ Arginine (Arg), cysteine (Cys), serine (Ser) and threonine (Thr) are totally destroyed but tryptophan (Trp) survives: use alkali if presence of tryptophan is suspected. Acidic conditions generally destroy fewer amino acids totally and so is preferred to alkaline hydrolysis. 38 19 Mild acid hydrolysis ▯ Conditions: 6M HCl, RT, several days. ▯ The amide bond between certain residues hydrolyse faster than others resulting in partial hydrolysis and the formation of peptide fragments. ▯ E.g. the side-chain functional group of aspartic acid (Asp) can be involved in hydrolysis (neighbouring group participation). ▯ This can also happen with glutamic acid (Glu), via a 6-ring anhydride, but to a lesser extent. O O O O OH O +/-H O + O H O +/-H O N OH H C N H 2 H3C N N 3 H N H3C N H H O H H O O H O + + H H O H2O O OH O OH H2N OH H C N N 3 H H O O 39 More reactions of peptides If a peptide ester is reduced with LiBH then 4ydrolysed with 6M acid, the C-terminus residue can be identified as an amino alcohol Ph O O O i, LiBH H H 4 Ph H2N N N ii, 6M HCl OH H2N N OMe OH H OH H N H 2 O O H2N 2 OH O O If a peptide is reacted with hydrazine then all the amino acids except the C-terminus residue will be isolated as the acid hydrazide O Ph H N O O Ph 2 NHNH H H 2 N N NH2NH 2 H2N N OH NHNH O NHNH2 H H N 2 H2N O O 2 H2N OH O O 40 20 End group analysis ▯ Sanger’s reagent ▯ Sanger’s reagent is fluoro-2,4-dinitrobenzene (FDNB) ▯ FDNB reacts with primary amine atoms to give acid-stable dinitrophenyl (DNP) derivatives ▯ It was developed to assist in the sequencing of peptides (determination of the primary structure) ▯ After total acid hydrolysis the DNP derivatized amino acid can be identified by comparison with standards. The a-DNP derivatized amino acid must correspond to the N-terminus of the peptide ▯ However the side-chain amine groups in lysine (Lys) and ornithine (Orn, whose side chain is one methylene shorter than lysine) react as well, but these can also be identified using chromatographic separation and by reference to standards ▯ An alternative to Sanger’s reagent is dansyl chloride which produces acid-stable sulfonamides 41 Sanger’s reagent and dansyl chloride nucleophilic aromatic Sanger’s F substitution H DNP reagent N R derivative +H2N R O2N NO2 O N NO coloured 2 2 yellow O H SO2Cl O S N R +H 2 R Absorbs strongly in dansyl NMe NMe the UV-Vis chloride 2 2 region Can detect mg -8 quantities (10 mol) 42 21 NH2 Ph O O i, FDNB H H ii, HCl, 6M, heat Mixture of amino acids N N for analysis (including H2N N OH H those tagged by FDNB) O O Cannot be N- NO2 terminus as only e-NH DNP O2N NO2 Ph 2 tagged OH N NO2 H OH O HN H N 2 4 OH O H2N OH H 2 O Must be O N-terminus as a-NH 2 is DNP tagged DNP-Phe a-tagged Lys(DNP) side chain tagged 43 Edman degradation ▯ The Sanger method is destructive and only determines the N- terminus residue of a peptide. ▯ Edman’s reagent (Ph-N=C=S) can be used to cleave sequentially the N-terminal residue of a peptide without the need for total hydrolysis. R 1 O S R1 O H H N Ph N Ph N C S H2N H N N N H H H O R2 O R2 6M HCl, rt Thiohydantoin R H+ O R 1 H 1 R1 N O HN N O H HN HN O + H2N Ph O R N N S 2 N S H S Ph R2 H Ph N The thiohydantoin can be isolated and compared to reference samples in order to identify the amino acid from the N terminus of the peptide. 44 22 Automated Peptide Sequencer The Edman process can be automated and used to determine the sequence of long peptides with 10s of amino acids. 45 Cyanogen bromide (CNBr) Cyanogen bromide selectively cleaves peptides on the C-side of methionine (Met) residues. This chemoselective reaction uses the nucleophilic sulfur atom and fast 5-exo cyclisation forming the five- membered ring (cf. faster hydrolysis at the C-side of Asp residues). R R 3 H 3 N H N R HN R N 1 H 1 H N O N▯C-Br N O N O N O H H O O S S Me Me CN R3 H R1 O R3 N H H H2O R1 N N + N H H O H2N N O N O O O H O 46 23 Enzymic cleavage of peptides ▯ Enzymes are protein catalysts used in a very wide range of biological reactions. ▯ Enzyme action may be mediated in a variety of ways: ▯ catalysis of phosphorylation of hydroxyl groups in serine (Ser) or tyrosine (Tyr) residues of other proteins ▯ cleavage of peptide (amide) bonds in other peptides or proteins. ▯ Some biological reactions may be very simple, for example digestion of proteins and carbohydrates (i.e. food) in the stomach. In this case, the hydrolysis of bonds is indiscriminate. ▯ In many cases, however, the action of an enzyme is highly selective, for example in the activation and deactivation of biologically active molecules such as hormones and neurotransmitters etc. 47 Renin-Angiotensin System and Hypertension Angiotensinogen Asp.Arg.Val.Tyr.Ile.His.Pro.Phe.His.Leu.Leu.Val.Tyr.Ser…(protein) Renin Angiotensin I Asp.Arg.Val.Tyr.Ile.His.Pro.Phe.His.Leu Angiotensin converting enzyme Angiotensin II Asp.Arg.Val.Tyr.Ile.His.Pro.Phe + His.Leu Angiotensinase degradation products 48 24 The Renin-Angiotensin Pathway ▯ The protein angiotensinogen, produced in the liver, is cleaved by the action of the enzyme renin to give angiotensin I, a decapeptide. ▯ Angiotensin I is converted to angiotensin II, an octapeptide, by the action of angiotensin converting enzyme or ACE. ▯ ACE cleaves a specific bond between the Phe-His residue so removing a dipeptide unit from the C-terminus of angiotensin I. ▯ Angiotensin II interacts with receptors on the surface of blood vessel cells. This mediates vasoconstriction to increase the blood flow when needed, but also causes fluid retention which together can result in high blood pressure (hypertension). The action of angiotensin II is moderated by angiotensinase an enzyme that degrades angiotensin II in to smaller inactive fragments. 49 Useful Enzymes Carboxypeptidase ▯ This (type of) enzyme sequentially cleaves amino acids from the C-terminus of a peptide chain as long as the a-2O H group is underivatized (unblocked). ▯ The rate of reaction for each residue is different, but is sufficiently slow that quenching the reaction after fixed times and quantifying the liberated amino acids allows the first two or three residues of the peptide to be determined. Aminopeptidase ▯ This (type of) enzyme sequentially cleaves amino acids from the N-terminus of a peptide chain as long as the a2NH group is underivatized. The rate is very rapid and cannot be used in partial sequencing as with carboxypeptidase. 50 25 Enzymes (cont.) ▯ Chymotrypsin ▯ Chymotrypsin recognizes residues with aromatic side chains. It cleaves on the carboxyl side of phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) residues ..AAn.Phe▯AA n+2... …AA nTyr▯AA n+2… …AA .Tnp▯AA n+2.. ▯ Trypsin ▯ Trypsin recognizes residues with basic side chains. It cleaves on the carboxyl side of arginine (Arg) and lysine (Lys) residues (but not histidine which is insufficiently basic) ….. AA .Lys▯AA ….. ….. AA .Arg▯ AA ….. n n+2 n n+2 51 Enzymes (cont.) ▯ Thermolysin ▯ Thermolysin recognizes residues with hydrophobic side chains. It cleaves on the amino side of phenylalanine (Phe), tryptophan and also leucine (Leu) residues. … AA ▯Phe.AA …. .... AA ▯Trp.AA …. n n+2 1 n+2 .…AA ▯neu.AA n+2…. ▯ These enzymes will normally not ‘work’ where D-amino acids are involved, as will also happen, in many cases, when proline (Pro) is present at nA or n+2 52 26 Polypeptide Sequencing 53 Introduction ▯ How do you determine the primary sequence of amino acids in a biologically interesting polypeptide? ▯ We need to use a combination of experiments and some logic! ▯ Luteinizing hormone-releasing hormone (LH-RH) was isolated in a few tens of μg from 1,000s of pig brains, yet was still successfully sequenced. What follows is a summary of the chemical and enzymatic reactions encountered previously in this course. 54 27 Total acid hydrolysis ▯ Heating a peptide with 6M HCl hydrolyses the peptide into its constituent amino acids. ▯ Amino acids can then be separated using various chromatographic techniques. ▯ The total number and distribution of amino acids can be determined. HOWEVER: ▯ Tryptophan (Trp) is destroyed by strong acid. ▯ Asparagine (Asn) and Glutamine (Gln) have amidated ω- carboxylic acid groups, which are hydrolysed by strong acid. Thus Asn and Gln residues appear as Asp and Glu residues after total acid hydrolysis. 55 Partial Hydrolysis ▯ Partial hydrolysis can be achieved with non-selective enzymes, e.g. papain. ▯ Peptide fragments are formed at random, producing for example di-, tri- and tetrapeptides. ▯ Weaker acid or 6M at lower temperatures does the same job. ▯ These fragments can be extremely useful in “filling in the gaps” with partially sequenced peptides (see later). 56 28 Chemical Techniques ▯ Cyanogen Bromide (CNBr) ▯ Cleaves on the C-side of methionine residues, Met▯Xaa ▯ Sanger’s reagent (fluorodinitrobenzene, FDNB) ▯ ‘Tags’ free 2H groups before total acid hydrolysis. Dinitrophenyl (DNP) tagged residues can be compared with reference samples (e.g. DNP-Gly) ▯ Edman degradation (phenyl isothiocyanate) ▯ Cleaves N-terminus as thiohydantoin derivative which can be compared with reference samples. 57 Enzymes Enzymes digest (cleave) peptides with a high degree of specificity. ▯ Trypsin ▯ Cleaves on the C-side of basic residues - Arginine and Lysine: Xaa.Arg▯Yaa Xaa.Lys▯Yaa ▯ Chymotrypsin ▯ Cleaves on the C-side of aromatic residues - Phenylalanine, Tyrosine and Tryptophan Xaa.Phe▯Yaa Xaa.Tyr▯Yaa Xaa.Trp▯Yaa ▯ Carboxypeptidase ▯ Sequentially cleaves the C-terminus residue from a peptide as long as it is the free a-acid (e.g. not an amide or ester). Reaction rates vary for different amino acids so the enzyme can only be used to determine a few residues before things get confusing. 58 29 Example 1 Consider the heptapeptide Val.Leu.Lys.Phe.Ala.Glu.Ala ▯ What would you expect to get if you treated the peptide with chymotrypsin? ▯ ANSWER: Val.Leu.Lys.Phe and Ala.Glu.Ala What we in fact get in reality is (Leu, Lys, Phe, Val) and (Ala ,Glu) after acid hydrolysis of each fragment. However we can ‘place’ the Phe-containing fragment at the N-terminus and the Phe at position 4 because of the known enzyme specificity. Chymotrypsin cleaves on the C-side of aromatic residues. ▯ What would you expect to get if you treated the peptide with trypsin? ▯ ANSWER: Val.Leu.Lys and Phe.Ala.Glu.Ala What we get are (Leu, Lys, Val) and (Ala ,Glu, Phe) but we can place the Lys-containing fragment at the N-terminus and the Lys at position 3. Trypsin cleaves on the C-side of basic residues 59 Example 1 (cont.) so far we have: (Leu,Val).Lys.Phe.(2la ,Glu) ▯ Treatment of the heptapeptide with Sanger’s reagent, followed by total acid hydrolysis gives DNP-Val. So Val is at position 1 and therefore by deduction Leu must be at position 2. ▯ Action of carboxypeptidase releases Ala first, thus giving the C- terminus residue. ▯ Treatment of the tripeptide produced by the action of chymotrypsin with Sanger’s reagent with gives DNP-Ala, thus placing Ala at position 5 and therefore Glu must be at 6. Sequence is: Val.Leu.Lys.Phe.Ala.Glu.Ala Five experiments used to sequence the heptapeptide 60 30 The Principle of Overlap ▯ A peptide containing (Ala,Gly,Pro,Ser) gives three dipeptides upon partial acid hydrolysis. After sequencing the dipeptides we get: Ser.Ala Ala.Gly Pro.Ser ▯ What is the sequence of the original peptide? Pro.Ser Ser.Ala Ala.Gly ▯ ANSWER: Pro.Ser.Ala.Gly 61 Example 2 ▯ A peptide containing (Ala,Arg,Cys,Leu,Val) gives four dipeptides upon partial acid hydrolysis. After sequencing the dipeptides we get: Cys.Arg Ala.Cys Leu.Ala Arg.Val ▯ What is the sequence of the peptide? Leu.Ala Ala.Cys Cys.Arg Arg.Val ▯ ANSWER: Leu.Ala.Cys.Arg.Val ‘Lone’ amino acids are extremely useful marker residues. 62 31 Deductions ▯ Partial acid or non-specific enzymic hydrolysis results in random production of smaller peptide fragments. This can be useful towards the end of sequencing particularly if the fragments contain unique or infrequently occurring amino acids. ▯ For example, if a peptide only contains two Gly residues and only one type of tripeptide wit2 Gly is isolated upon partial hydrolysis then the only scenarios that can give rise to this are: ▯ …..Gly.Xaa.Gly….. in the middle of a peptide ▯ Gly.Gly.Xaa….. or Gly.Xaa.Gly….. at N-terminus ▯ …..Xaa.Gly.Gly or …..Gly.Xaa.Gly at C-terminus 63 Problem 1 A polypeptide is subjected to the following digestion procedures and the fragments were sequenced. What is the sequence of the original peptide? Cyanogen bromide Trypsin digestion A Asp.Ile.Lys.Gln.Met D Gln.Met.Lys B Lys.Phe.Ala.Met E Gly.Met.Asp.Ile.Lys C Tyr.Arg.Gly.Met F Phe.Ala.Met.Lys and Lys G Tyr.Arg ▯ Lys must be at C-terminus from CNBr experiment, but overlap could be with fragment D or F. ▯ Look for alternating overlap of fragments from each experiment. How about C and G ? These both start with the same residues. 64 32 Problem 1 (cont.) C Tyr.Arg.Gly.Met G Tyr.Arg E Gly.Met.Asp.Ile.Lys A Asp.Ile.Lys.Gln.Met D Gln.Met.Lys B Lys.Phe.Ala.Met F Phe.Ala.Met.Lys Lys Sequence is: G E D F Tyr.Arg.Gly.Met.Asp.Ile.Lys.Gln.Met.Lys.Phe.Ala.Met.Lys C A B 65 Problem 2 A peptide was digested with enzymes and gave the following fragments. Trypsin digestion A Ser.Gln.Met.Thr.Glu.Arg B Asn.Ala.Trp.Leu.Lys C Ala.Pro.Asp Chymoptrypsin digestion D Leu.Lys.Ser.Gln.Met.Thr.Glu.Arg.Ala.Pro.Asp E Asn.Ala.Trp 66 33 Problem 2 (cont.) C must be the C-terminus as it has no basic residue, so (A,B)▯C C Ala.Pro.Asp D Leu.Lys.Ser.Gln.Met.Thr.Glu.Arg.Ala.Pro.Asp A Ser.Gln.Met.Thr.Glu.Arg B Asn.Ala.Trp.Leu.Lys E Asn.Ala.Trp Sequence is: Asn.Ala.Trp.Leu.Lys.Ser.Gln.Met.Thr.Glu.Arg.Ala.Pro.Asp 67 Bradykinin Bradykinin, A, is a nonapeptide released by blood plasma globulins in response to a wasp sting. It is a potent pain-causing agent. Total acid hydrolysis of A gives (Arg , Gly, Phe , Pro , Ser) 2 2 3 Chymotrypsin digestion gives: Partial acid hydrolysis gives: peptide B (Arg,Gly,Phe,Pro ) Phe.Ser 2 peptide C (Phe,Pro,Ser) Pro.Gly.Phe and free Arg Pro.Pro Ser.Pro.Phe Trypsin digestion gives: Phe.Arg peptide D (Arg,Pr3 ,Gly,Ph2 ,Ser) Arg.Pro and free Arg 68 34 Bradykinin (cont.) (Arg, Arg, Gly, Phe, Phe, Pro, Pro, Pro, Ser) ▯ From the chymotrypsin digestion Arg must be at the C-terminus ▯ Phe must be at positions 5 and 8 or 3 and 8 (depending on B/C order) due to enzyme specificity. ▯ From the trypsin digestion Arg is at the N-terminus too. ▯ From the dipeptides Arg.Pro and Phe.Arg we must conclude Pro is at position 2 and Phe is at position 8 Arg.Pro __ __ __ __ __ Phe.Arg 1 2 3 4 5 6 7 8 9 69 Bradykinin (cont.) ▯ Di- and tri-peptides from partial hydrolysis: Phe.Ser ; Pro.Gly.Phe ; Pro.Pro ; Ser.Pro.Phe ; Phe.Arg ; Arg.Pro ▯ There is only one Ser residue so we must have Phe.Ser.Pro.Phe from overlapping fragments. ▯ The Pro.Pro dipeptide must overlap with the Pro.Gly.Phe tripeptide as the other Pro wedged between Ser and Phe so we are left with: Phe.Ser.Pro.Phe and Pro.Pro.Gly.Phe Arg.Pro __ __ __ __ __ Phe.Arg 1 2 3 4 5 6 7 8 9 ▯ Bradykinin must bArg.Pro.Pro.Gly.Phe.Ser.Pro.Phe.Arg 1 2 3 4 5 6 7 8 9 70 35 Phylloxin (June 2001) Phylloxin, A, is a peptide isolated from the skin of the hylid frogs belonging to the genus Phyllomedusinae. Total acid hydrolysis results in the isolation of the following amino acids: A (Ala, Cys, Gl3 , G2u ,2Ile , Leu, Lys, Met, Ph3, Se2 , Tyr ) Use the information to establish the sequence of amino acids in phylloxin. Trypsin cleaves on the C-side of basic residue, Arg↓Xaa and Lys↓Xaa CNBr cleaves on the C-side of methionine, Met↓Xaa Chymotrypsin cleaves on the C-side of aromatic residues Tyr↓Xaa and Phe↓Xaa Fluorodinitobenzene and acid hydrolysis gives N-terminus DNP-Xaa derivative Edman degradation sequentially cleavesN-terminus residues. Note well the single (‘marker’) residues 71 Phylloxin (cont.) Trypsin cleaves A to give two peptides. ▯ Only one Lys and no Arg, so C must be at the N-terminus. ▯ FDNP gives DNP-Ile at N-terminus of B C B (Gly,Met,Ser,Tyr).Lys↓Ile(Ala,Cys,Gly ,Glu ,Ile,Leu,Phe,Ser ,Tyr) 2 2 2 5 6 ▯ CNBr gives two peptides; only one Met present so tripeptide E must be at the N-terminus with Met at position 3. E F (Xaa,Yaa)Met↓(Zaa).Lys.Ile(Ala,Cys,Gl2 ,Gl2 ,Ile,Leu,Phe,2er ,Tyr) 3 5 6 72 36 Phylloxin (cont.) Chymotrypsin cleaves A to give three peptides and free Phe. ▯ G must be at the C-terminus as it contains no aromatic residue. ▯ F contains the only Met and Lys so must be in the middle and must end in Tyr. ▯ H must be at the N-terminus: it contains Tyr and Gly [c.f. fragments C and E] therefore (Gly,Tyr) becomes Gly.Tyr, and with Ser fall into positions 1,2 and 4 respectively. ▯ Phe must therefore fall into position 13. H F G Gly.Tyr↓Met.Ser.Lys.Ile(Ala2Gly ,Ile,Ser).Tyr↓Phe↓(2ys,Glu ,Leu,Ser) 1 2 3 4 5 6 12 13 73 Phylloxin (cont.) Ta


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