University:
Boston University
Course:
GRS CH 623 | Chemical Biology
Academic year:

2021
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256

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21
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iluhanbt81

Amino Acids: Classification • The 20 amino acids found in proteins can be placed in five families based on the physical and chemical properties of their R groups: • Hydrophobic, aliphatic (6) Gavlip family • Hydrophobic, aromatic (3) PTT family • Special (hydrophobic/hydrophilic)(2) MC family • Hydrophilic, polar (4) • Hydrophilic, charged (5) 1 Amino Acids: Classification Hydrophilic, polar These amino acids side chains can form hydrogen bonds. Qnst family Gln Name Amides 3-letter Glutamine Asparagine Serine Threonine ✓ Gln Asn Ser Thr Year 1-letter discovered Q N S T Ser Asn 1883 1806 1865 1935 % abundance in proteins 4 4 7 6 Alcohols Thr Structure mnemonic device NOTES Glx; gets hydrolyzed to Glu Amide of Glu First isolated from asparagus Asx; gets hydrolyzed to Asp Amide of Asp Isolated from Sericin, polar hydroxyl+Ala cousin of Ala Two chiral centers (L & D) Me+Ser Amino Acids: Classification • The 20 amino acids found in proteins can be placed in five families based on the physical and chemical properties of their R groups: • Hydrophobic, aliphatic (6) Gavlip family • Hydrophobic, aromatic (3) PTT family • Special (hydrophobic/hydrophilic)(2) MC family • Hydrophilic, polar (4) • Hydrophilic, charged (5) Qnst family 2 Hydrophilic, Charged Amino Acids: Classification Asp Lys Negative Name 3-letter 1-letter Aspartate Glutamate Asp Glu D E % Year abundance discovered in proteins 1868 1866 5 7 His Positive NOTES a-amino-succinate; Most acidic a-amino-glutarate pKa Structure mnemonic device 3.7 Ala+carboxyl 4.3 Ala+acetate Amino Acids: Naming Dicarboxylic Acids 3 Hydrophilic, Charged Amino Acids: Classification a a b b – g R HN g – HN R d d e HN R R – Asp – HN As the electrons creating the double bond become de-localized, the positive charge also becomes delocalized Lys Negative Name 3-letter 1-letter Aspartate Glutamate Lysine Arginine Histidine ✓ ✓ ✓ Asp Glu Lys Arg His % Year abundance discovered in proteins D E K R H Hydrophilic, Charged 1868 1866 1889 1886 1896 5 7 6 5 1 His Positive NOTES pKa a-amino-succinate; Structure mnemonic device 3.7 Ala+carboxyl 4.3 Ala+acetate Only “bis” amino acid 10.5 e-amino 12.5 d-guanidino Most basic Only physiological 6.0 Ala+imidazole ionization Most acidic a-amino-glutarate Amino Acids: Classification a a b b g g d d e Dekrh family Asp Lys Negative Name 3-letter 1-letter Aspartate Glutamate Lysine Arginine Histidine ✓ ✓ ✓ Asp Glu Lys Arg His D E K R H % Year abundance discovered in proteins 1868 1866 1889 1886 1896 5 7 6 5 1 His Positive NOTES a-amino-succinate; pKa Structure mnemonic device 3.7 Ala+carboxyl 4.3 Ala+acetate Only “bis” amino acid 10.5 e-amino 12.5 d-guanidino Most basic Only physiological 6.0 Ala+imidazole ionization Most acidic a-amino-glutarate 4 Amino Acids: Classification • The 20 amino acids found in proteins can be placed in five families based on the physical and chemical properties of their R groups: Name • Hydrophobic, aliphatic (6) Gavlip family • Hydrophobic, aromatic (3) PTT family • Special (hydrophobic/hydrophilic)(2) MC family • Hydrophilic, polar (4) • Hydrophilic, charged (5) 3-letter 1-letter Glycine Gly Alanine Ala Valine ✓ Val Leucine✓ Leu Isoleucine✓ Ile Proline Pro Phenylalanine✓Phe Tyrosine Tyr Tryptophan ✓ Trp Methionine ✓ Met Cysteine Cys Glutamine Gln Asparagine Asn Serine Ser Threonine ✓ Thr Aspartate Glu Glutamate Asp Lysine ✓ Lys Arginine ✓ Arg Histidine ✓ His G A V L I P F Y W M C Q N S T D E K R H Qnst family Structure % Year abundance discovered in proteins 1820 1888 1856 1819 1904 1901 1879 1846 1901 1922 1899 1883 1806 1865 1935 1868 1866 1889 1886 1896 7 8 7 10 6 5 4 3 1 2 2 4 4 7 6 5 7 6 5 1 Dekrh family NOTES pKa mnemonic device Smallest, not chiral Foundational for ~10 other AA isopropyl H Methyl V-shaped Most abundant, dominant Ala + Val Two chiral centers (L & D) Val + Me Only imino acid (2° amine); special bonds in proteins; is modified by OH 5-membered ring; same # as Val; 3C aromatic aromatic, can ionize; amphipathic Phenyl+Ala 10.1 p-phenol+Ala aromatic & fluorescent; least abundant Indole+Ala Most like straight-chain aliphatic Ala+Me/ether can ionize; nucleophile 10.5 Ala+SH Glx; gets hydrolyzed to Glu Amide of Glu First isolated from asparagus Asx; gets hydrolyzed to Asp Isolated from Sericin, polar cousin of Ala Two chiral centers (L & D) a-amino-succinate; Most acidic a-amino-glutarate Only “bis” amino acid Most basic Only physiological ionization Amide of Asp hydroxyl+Ala Me+Ser 3.7 Ala+carboxyl 4.3 Ala+acetate 10.5 e-amino 12.5 d-guanidino 6.0 Ala+imidazole 5 Other Cool Amino Acids a b g d 3 3 3 D-homoserine D-ornithine (Orn) L-thyroxine (thyroid hormone) ⊕ 3 Modified Amino Acids Found in Proteins •Not incorporated by ribosomes Reversible: -except for selenocysteine •Arise by posttranslational modifications of proteins •Reversible modifications, especially phosphorylation, are important in regulation and signaling. 6 Amino Acids Polymerize by Peptide Bonds hydrolysis condensation • Using full amino acid names: – serylglycyltyrosylalanylleucine • Using the three-letter code abbreviation: – Ser-Gly-Tyr-Ala-Leu • For longer peptides (like proteins) the one-letter is used: – SGYAL Peptide Bond N-term C-term In a protein, the Ionizable Side Chains have altered pKa values as AA DpKa 2.3 +0.8 3.7 4.3 +0.7 +0.1 6.0 +0.5 9.6 –1.6 10.5 –2.0 10.1 –0.1 10.5 –0.5 12.5 –0.5 7 Chemical Environment Affects pKa Values EXAMPLE: a-carboxy group is much more acidic than in carboxylic acids. IN PROTEINS, the environment can be much different than in bulk solution, and pKa values can change by several orders of magnitude. Amino Acids Can Act as Buffers Question: For one mole of completely protonated Gly, how many moles of base are required to titrate all the protons? 2 Cation à Zwitterion à Anion Question: What does the curve of pH versus moles of base look like (i.e., the titration curve)? Curved Amino acids with uncharged side chains, such as glycine, have two pKa values: •The pKa of the a-carboxyl group is 2.34. •The pKa of the a-amino group is 9.6. Buffer Regions As buffers prevent change in pH close to the pKa, glycine can act as a buffer in two pH ranges. Question: Why is this not linear? 8 Amino Acids Can Act as Buffers Question: Why is this not linear? Henderson- Hasselbalch Eqn! % dissoc iated [A–]/[HA] pH 50 1/1 pKa 10 10/90 pKa– 0.95 1 1/99 pKa–2 0.1 1/999 pKa–3 90 90/10 pKa+ 0.95 99 99/1 pKa+2 99.9 999/1 pKa+3 5 4 3 2 1 Amino Acids Can Act as Buffers •Those amino acids with Ionizable side chains can be also titrated. His •Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units. Asp 9 Amino Acids Can Act as Buffers •Those amino acids with Ionizable side chains can be also titrated. His •Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units. Asp Amino Acids Carry a Net Charge of Zero at a Specific pH value (the pI) • The Isoelectric Point (equivalence point, pI) is the pH value where the net charge is ZERO. • Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups. • The exact value is the average of the two pKa values forming or titrating the zwitterion. • At the pH equal to the pI: – AA is least soluble in water. pI = pK1 + pK 2 2 – AA does not migrate in electric field. – AA does not bind well to other charged media/compounds 10 How to Calculate pI What is the pI of glycine? • Identify the zwitterion (species that carries a net charge of zero). • Identify the pKa value for the reaction that protonates the zwitterion. • Identify the pKa value for the reaction that titrates a proton from the zwitterion. • Take the average of these two pKa values. pK1 + pK 2 pI = 2 pI = (2.3 + 9.6)/2 pI = 5.95 pI = 5.95 How to Calculate the pI When the His Side Chain Is Ionizable • Identify species that carries a net zero charge. pI = 7.6 • Identify the pKa value for the reaction that protonates the zwitterion. For His this occurs on the R-group (pKR). • Identify the pKa value for the reaction that titrates the next proton from the zwitterion. For His this is the a-amino group (pKNH ). pK1 + pK 2 2 pI = 2 • Take the average of these two pKa values. pI = (6.0 + 9.2)/2 What is the pI of histidine? pI = 7.6 11 How to Calculate the pI of a peptide !Es#mate(the(pI(value(of(the(following(hexapep#de:( ! Phe9Lys9Asp9Cys9Thr9Tyr( ! Step!1:!Determine!the!total!posi2ve!charge!on!the!pep2de!when!all!acidic!and!basic! groups!are!fully!protonated!(at!low!pH).!! Step!2:!Determine!the!total!nega2ve!charge!on!the!pep2de!when!all!the!groups!are! 2trated!(at!high!pH).! Step!3:!!List!the!pKa!values!of!all!acidic!and!basic!groups!in!order!from!lowest!(pKa1)!to! highest.!! Step!4:!Calculate!the!pI!as!the!average!of!the!values!for!pKa!value!of!the!proton! dissocia2on!forming!a!neutral!species!from!a!+1!species,!and!pKa!value!of!the! proton!dissocia2on!forming!a!–1!species!from!the!neutral!species.! ! So!for!this!pep2de!! ! Step!1:!!charge!when!fully!protonated!+2! ! Step!2:!!charge!when!fully!deIprotonated!I4! ! !values!are:!! Step!3:!!pK a 9.0(NIterm),!10.5(Lys),!3.9(Asp),!8.4!(Cys),!10.5(Tyr),!3.5(CIterm)! List!from!lowest!to!highest!!! ! !!!!!!pKa!!!!!!!!!!!!!!!!!!!!!!!!!!!3.5!!!!3.9!!!!!8.4!!!!!9.0!!!!!!10.5!!!!!!10.5!!! Charges!!!!!!!!!!!!!!!!!!!+2!!!!+1!!!!0!!!!!I1!!!!!I2!!!!!I3!!!!!!!I4! ! Step!4:!!The!pI!is!!(3.9!+!8.4)/2!=!6.2! ! Proteins and their pI Values pH at: Cation à Zwitterion à Anion pI • IN GENERAL, if you take the % abundance of acidic and basic residues (Glu+Asp) and (Lys+Arg), you have ~12 and ~11%. • So, there are slightly more acidic residues than there are basic residues. • Half way between the most basic of these acidic residues lies the pI values for most proteins; most are below the average of 4.3 (Glu) + 10.5 (Lys) ÷ 2 = 7.4. • Therefore, given that there is 1% more (Glu+Asp) than (Lys+Arg), most proteins are slightly more acidic than physiological pH. Ø That doesn’t mean there are not many proteins that are very acidic (pI values <<7.4; negatively charged at pH 7.4): – – – – Transcription factors Pepsin Ovalbumin Serum albumin • Like AA, for proteins, at the ØOr, very basic (pI values >>7.4; positively charged at pH 7.4): – – – – Cytochrome c Lysozyme Histones Ribosomal proteins pH equal to the pI: – Protein is least soluble in water, could precipitate. – Protein does not migrate in electric field. 12 OUTLINE • Reading: Amino Acids – Definition, Structure, and Properties • The 4 S’s • Common Properties • Five Classes • Homework #5 – – – – – Hydrophobic–aliphatic [6] Hydrophobic–aromatic [3] Special–sulfur [2] Hydrophilic–polar [4] Hydrophilic–charged [5] Ch3; 76–82, 87–89 NEXT • Reading: • Ch1; Fig 7 Ch9; 313–314 Homework #6 Ch3; 83–87, 89–90 • Other amino acids • Linking amino acids • Acid/base properties – Titrations – Isoelectric point • Electrophoresis Protein Purification Electrophoresis for Protein Analysis • Electrophoresis is the migration of molecules in an electric field. • Electrophoresis is one of the most commonly used analytical scale separation techniques – The electric field pulls proteins according to their charge. • Gel electrophoresis adds a solid support in which the separation occurs. The gel matrix hinders mobility of proteins according to their size and shape. – The commonly used gels are either polyacrylamide (proteins) or agarose (nucleic acids). – separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE. • For proteins to separate, they have to have a charge. 13 Electrophoresis for Protein Analysis Gels Agarose Polyacrylamide Electrophoresis for Protein Analysis Gels Polyacrylamide 14 Electrophoresis for Protein Analysis • For proteins to separate, they have to have a charge. • The charge on a protein will depend on the pH. But once they are moving, what does the velocity depend on? Electric field Velocity = E•z charge on the protein f friction f ∝ mass, shape, viscosity of media f = 6∏ • " • r Velocity = where: " = coefficient of viscosity r = Stokes radius (mass and shape) [this is from actual radius and specific volume (cm3/g)] E z • 6∏ • " r This is essentially the charge:mass ratio if all proteins are roughly the same shape (globular). Electrophoresis for Protein Analysis • For proteins to separate, they have to have a charge. • pH and pI dependence: o At pH near the pI, not much movement o At pH below the pI, proton concentration is higher, so charge becomes positive ⊕ o At pH above the pI, protons will be titrated off, so the charge will become ⊝ Recall: 15 Isoelectric Focusing Takes advantage of the pI differences in Proteins for Separation Can also be used to determine the pI values Isoelectric Focusing Velocity = E z • 6∏ • ! r Ampholytes are highly charged small MW polymer with variable pI values. Due to their high z/r, they migrate rapidly setting up a buffered pH gradient. Once they reach the pH equal to their individual pI values, they STOP migrating, thus creating an immobilized pH gradient. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS PAGE) Separates Proteins by Molecular Weight • SDS – sodium dodecyl sulfate – a detergent Velocity = E z • 6∏ • ! r • SDS micelles bind to proteins and facilitate unfolding. – SDS gives all proteins a uniformly negative charge and shape (micelle) – The native shape is perturbed; de-natured. – SDS binds proteins at a constant ratio of mass (1.4g SDS/g protein), coating them with a negative charge. – So much charge is added that all proteins have the same charge:mass ratio, and the rate of movement will only depend on the sieving properties of the gel: small proteins will move farther. 16 SDS PAGE Separates Proteins by Molecular Weight SDS-PAGE Used to detect purity or purification SDS PAGE Can Be Used to Calculate the Molecular Weight of a Protein Used to determine the molecular weight 17 SDS-PAGE + Isoelectric Focusing Can Separate nearly ALL the Proteins in E. coli size charge Used to separate all proteins in a cell: proteomics Protein Purification 18 Protein Purification Proteins are separated from each other (along with other macromolecules) due to the vast variability they have. The basis of the separation can be put into 4 categories: • Size, shape, density • Charge • Solubility • Binding characteristics } } } Hydrodynamic properties Chemical properties Biological properties Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab 19 Before you can separate “your favorite protein (YFP)” from all the thousands of others, you need a way to “see” it.…..... An Assay! • enzymes ➟ activity assay • binding proteins ➟ binding assay • other proteins ➟ immunoblot or ELISA How do you monitor the purification? • Specific Activity = Activity of YFP Total protein • Specific Activity = Activity of YFP Total protein How do you measure Specific Activity? What is the Yield? blue (of red proteins?) blue blue 20 What is the specific activity? From [activity] (U/mL) From [protein] (mg/mL) Purification of a hypothetical protein -If protein was “pure” after step #5, what would the Specific Activity be after you performed a step #6? -What is the Yield? 21

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