Amino Acids, Peptides, and Proteins PDF

3 Amino Acids, Peptides, and Proteins © 2021 Macmillan Learning 3.1 Amino Acids  Amino Acids Share Common Structural Features α carbon and four substituents α carbon is the chiral center tetrahedral  Amino Acid Substituents four substituents:...

3 Amino Acids, Peptides, and Proteins © 2021 Macmillan Learning 3.1 Amino Acids  Amino Acids Share Common Structural Features α carbon and four substituents α carbon is the chiral center tetrahedral  Amino Acid Substituents four substituents: – a carboxyl group – an amino group – a hydrogen atom – an R group (a side chain unique to each amino acid) glycine has a second hydrogen atom instead of an R group  The Amino Acid Residues in Proteins are L Stereoisomers two possible stereoisomers = enantiomers optically active D, L system specifies absolute configuration Amino Acids Can Be Classified by R Group five main classes: – nonpolar, aliphatic (7) – aromatic (3) – polar, uncharged (5) – positively charged (3) – negatively charged (2)  Answer Which amino acid has three pKa values? E. cysteine Cysteine has three pKa values: pK1 (—COOH): 1.96 pK2 (—NH3+): 10.28 pK3 (R group): 8.18 Nonpolar, aliphatic R groups the hydrophobic effect stabilizes protein structure Aromatic R Groups R groups absorb UV light at 270–280 nm can contribute to the hydrophobic effect Polar, Uncharged R Groups R groups can form hydrogen bonds cysteine can form disulfide bonds Positively Charged R Groups have significant positive charge at pH 7.0 Negatively Charged R Groups have a net negative charge at pH 7.0 Amino Acids Can Act as Acids or Bases amino groups, carboxyl groups, and ionizable R groups = weak acids and bases zwitterion occurs at neutral pH  Titration of Amino Acids cation ⇌ zwitterion ⇌ anion —COOH has an acidic pKa (pK1) —NH3+ has a basic pKa (pK2) the pH at which the net electric charge is zero is the isoelectric point (pI)  Isoelectric Point, pI for amino acids without ionizable side chains, the isoelectric point (pI) is: pK1 + pK 2 pI = 2 pH = pI = net charge is zero (amino acid least soluble in water, does not migrate in electric field) pH > pI = net negative change pH < pI = net positive charge  Peptides Are Chains of Amino Acids peptide bond: – covalent – formed through condensation – broken through hydrolysis  Peptide Types by the Number dipeptide = 2 amino acids, 1 peptide bond tripeptide = 3 amino acids, 2 peptide bonds oligopeptide = a few amino acids polypeptide = many amino acids, molecular weight < 10 kDa protein = thousands of amino acids, molecular weight > 10 kDa  Peptide Terminals numbering (and naming) starts from the amino-terminal residue (N-terminal) N-terminal C-terminal  Naming Peptides full amino acid names: serylglycyltyrosylalanylleucine three-letter code abbreviations: Ser–Gly–Tyr–Ala–Leu one-letter code abbreviation: SGYAL  Answer Which statement is correct about peptides? B. Peptides have their amino acid sequences written from the N-terminus. Peptides are named beginning with the amino- terminal (N-terminal) residue. Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes and Compositions Table 3-2 Molecular Data on Some Proteins length of Number of naturally Molecular Number of polypeptide Protein weight residues chains occurring Cytochrome c 12,400 104 1 peptides = 2 (human) to many Myoglobin (equine 16,700 153 1 thousands of heart) Chymotrypsin (bovine 25,200 241 3 amino acid pancreas) residues Hemoglobin (human) 64,500 574 4 Hexokinase (yeast) 107,900 972 2 RNS polymerase (E. 450,00 4,158 5 coli) Glutamine synthetase 619,000 5,628 12 (E. coli) Titin (human) 2,993,000 26,926 1  Peptide Subunits multisubunit protein = 2+ polypeptides associated noncovalently oligomeric protein = at least 2 identical subunits – identical units = protomers  Amino Acid Composition of Proteins Table 3-3 Amino Acid Composition of Two Proteins Bovine cytochrome c: Bovine Bovine Number of cytochrome c: chymotrypsinogen: Bovine residues per percentage of Number of residues per chymotrypsinogen: Amino Acid molecule total molecule Percentage of total amino acid Ala Arg 6 2 6 2 22 4 1.6 9 composition Asn 5 5 14 5.7 is highly Asp 3 3 9 3.7 variable Cys 2 2 10 4 Gln 3 3 10 4 Glu 9 9 5 2 Gly 14 13 23 9.4 His 3 3 2 0.8 Ile 6 6 10 4 Leu 6 6 19 7.8 Lys 18 17 14 5.7 Met 2 2 2 0.8 Phe 4 4 6 2.4 Pro 4 4 9 3.7 Ser 1 1 28 11.4 Thr 8 8 23 9.4 Trp 1 1 8 3.3 Tyr 4 4 4 1.6 Val 3 3 23 9.4 Total 104 102 245 99.7  Estimating the Number of Amino Acid Residues number of residues = molecular weight/110 average molecular weight of amino acid = ~128 molecule of water removed to form peptide bond = 18 128 – 18 = 110  Some Proteins Contain Chemical Groups Other Than Amino Acids conjugated proteins = Table 3-4 Conjugated Proteins contain permanently Class Prosthetic group Example associated chemical Lipoproteins Lipids β1-Lipoprotein of blood components (Fig. 17-2) – non–amino acid part Glycoproteins Carbohydrates Immunoglobulin G (Fig. 5-20) = prosthetic group Phosphoproteins Phosphate groups Glycogen phosphorylase (Fig. 6-39) lipoproteins contain Hemoproteins Heme (iron porphyrin) Hemoglobin (Figs 5-8 to 5-11) lipids Flavoproteins Flavin nucleotides Succinate dehydrogenase (Fig. 19-9) glycoproteins contain Metallproteins Iron Zinc Ferritin (Box 16-1) Alcohol dehrogenase sugars (Fig. 14-12) Calcium Calmodulin (Fig. 12-17) Molybdenum Dinitrogenase (Fig. 22-3) metalloproteins contain Copper Complex IV (Fig. 19-12) specific metals 3.3 Working with Proteins  Proteins Can Be Separated and Purified separated based on: – size – charge – binding properties – protein solubility Methods for Purifying Proteins first step = break open tissue or microbial cells – crude extract = releases proteins in solution second step = fractionation = separate proteins into fractions based on size or charge – “salting out” = lower solubility of proteins in salt to selectively precipitate proteins third step = dialysis = use semipermeable membrane to separate proteins from small solutes  Column Chromatography first step = buffered solution (mobile phase) migrates through porous solid material (solid phase) second step = buffered solution containing protein migrates through solid phase protein properties affect migration rates  Answer Which component is absolutely necessary for the purification of a protein? C. a means of detecting the protein To study a protein in detail, researchers must be able to separate it from other proteins in pure form and must have the techniques to determine its properties. Ion-Exchange Chromatography separates based on sign and magnitude of the net electric charge pH and concentration of free salt ions affect protein affinity uses bound charged groups: – cation exchangers – anion exchangers Size-Exclusion Chromatography also called gel filtration chromatography separates based on size large proteins emerge from the column before small proteins do  Affinity Chromatography separates based on binding affinity eluted by high concentration of salt or ligand  Answer Which protein would elute first from a gel filtration column? C. protein C, a homodimer with protomer Mr = 35,300 Size-exclusion chromatography, also called gel filtration, separates proteins according to size. In this method, large proteins emerge from the column sooner than small ones do.  Sequential Purification Steps Decrease Sample Size Table 3-5 A Hypothetical Purification Table for an Enzyme Fraction Specific volume Total Activity activity Procedure or step (mL) protein (mg) (units) (units/mg) 1. Crude cellular extract 1,400 10,000 100,000 10 2. Precipitation with ammonium sulfate 280 3,000 96,000 32 3. Ion-exchange chromatography 90 400 80,000 200 4. Size-exclusion chromatography80 80 100 60,000 600 5. Affinity chromatography 6 3 45,000 15,000 Note: All data represent the status of the sample after the designated procedure has been carried out. “Activity” and “specific activity” are defined on page 90. final specific activity: starting specific activity ratio = purification factor percentage of the final activity/starting activity = percent yield  Proteins Can Be Separated and Characterized by Electrophoresis electrophoresis = visualize and characterize purified proteins can be used to estimate: – number of different proteins in a mixture – degree of purity – isoelectric point – approximate molecular weight Electrophoresis for Protein Analysis uses cross-linked polymer polyacrylamide gels proteins migrate based on charge-to- mass ratio visualization = Coomassie blue dye binds to proteins https://www.youtube.com/watch?v=MILiO1XnuqQ 3.4 The Structure of Proteins: Primary Structure  Levels of Structure in Proteins four levels: – primary structure = covalent bonds linking amino acid residues in a polypeptide chain – secondary structure = recurring structural patterns – tertiary structure = 3D folding of polypeptide – quaternary structure = 2+ polypeptide subunits Protein Structure Is Studied Using Methods That Exploit Protein Chemistry Traditional protein sequencing techniques: – labeling proteins – breaking proteins into parts (protease) Structure at atomic resolution: X-ray Crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, Cryo-EM (electron microscopy)  Mass Spectrometry Provides Information on Molecular Mass, Amino Acid Sequence, and Entire Proteomes mass spectrometry = measure molecular mass with high accuracy – Can identify protein when coupled with proteases – Can sequence short peptides https://www.youtube.com/watch?v=M5UMCiWTeqA https://www.youtube.com/watch?v=v8EsEWwrJWs https://www.youtube.com/watch?v=L6MHSb7I820