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Prentice Hall c2002Chapter 31 Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and the Primary Structures of Proteins Copyright © 2006 Pearson.

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Presentation on theme: "Prentice Hall c2002Chapter 31 Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and the Primary Structures of Proteins Copyright © 2006 Pearson."— Presentation transcript:

1 Prentice Hall c2002Chapter 31 Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and the Primary Structures of Proteins Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry Rawn

2 Prentice Hall c2002Chapter 32 Chapter 3 - Amino Acids and the Primary Stucture of Proteins Important biological functions of proteins 1. Enzymes, the biochemical catalysts 2. Storage and transport of biochemical molecules 3. Physical cell support and shape (tubulin, actin, collagen) 4. Mechanical movement (flagella, mitosis, muscles) (continued)

3 Prentice Hall c2002Chapter 33 Functions of proteins (continued) 5. Decoding cell information (translation, regulation of gene expression) 6. Hormones or hormone receptors (regulation of cellular processes) 7. Other specialized functions (antibodies, toxins etc)

4 Prentice Hall c2002Chapter General Structure of Amino Acids Twenty common  -amino acids have carboxyl and amino groups bonded to the  -carbon atom A hydrogen atom and a side chain (R) are also attached to the  -carbon atom

5 Prentice Hall c2002Chapter 35 Zwitterionic form of amino acids Under normal cellular conditions amino acids are zwitterions (dipolar ions): Amino group = -NH 3 + Carboxyl group = -COO -

6 Prentice Hall c2002Chapter 36 Fig 3.1 Two representations of an amino acid at neutral pH (b) Ball-and stick model (a) Structure

7 Prentice Hall c2002Chapter 37 Stereochemistry Stereoisomers ( 立體異構物 )- compounds that have the same molecular formula but differ in the arrangement of atoms in space Enantiomers - nonsuperimposable mirror images Chiral carbons - have four different groups attached

8 Prentice Hall c2002Chapter 38 Stereochemistry of amino acids 19 of the 20 common amino acids have a chiral  -carbon atom (Gly does not) Threonine and isoleucine have 2 chiral carbons each (4 possible stereoisomers each) Mirror image pairs of amino acids are designated L (levo) and D (dextro) Proteins are assembled from L-amino acids (a few D-amino acids occur in nature)

9 Prentice Hall c2002Chapter 39 Fig 3.2 Mirror-image pairs of amino acids

10 Prentice Hall c2002Chapter 310 Assignment of configuration by the RS system (a) Assign a priority to each group attached to a chiral carbon based upon atomic mass priority (1 highest, 4 lowest) If two atoms are identical, move to the next atoms For double or triple bonds, count atom once for each bond (-CHO higher priority than -CH 2 OH) Priorities (low to high): -H, -CH 3, -C 6 H 5, -CH 2 OH, -CHO, -COOH, -NH 2, -NHR, -OH, -OR, -SH

11 Prentice Hall c2002Chapter 311 RS system (continued) (b) Orient the molecule with priority 4 pointing away (behind the chiral carbon). Trace path from highest priority to lowest priority (1, 2, 3, 4) (c) Clockwise path: absolute configuration R Counterclockwise path: absolute configuration S NOTE: All of the 19 common chiral L-amino acids except cysteine have the S configuration.

12 Prentice Hall c2002Chapter 312 Assignment of RS configuration

13 Prentice Hall c2002Chapter Structures of the 20 Common Amino Acids Fischer projections - horizontal bonds from a chiral center extend toward the viewer, vertical bonds extend away from the viewer Abbreviations can be one letter or three letters Amino acids are grouped by the properties of their side chains (R groups) Classes: Aliphatic, Aromatic, Sulfur-containing, Alcohols, Bases, Acids and Amides

14 Prentice Hall c2002Chapter 314 A. Aliphatic R Groups Glycine (Gly, G) - the  -carbon is not chiral since there are two H’s attached (R=H) Four amino acids have saturated side chains: Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L)Isoleucine (Ile, I) Proline (Pro, P) 3-carbon chain connects  -C and N

15 Prentice Hall c2002Chapter 315 Four aliphatic amino acid structures

16 Prentice Hall c2002Chapter 316 Fig 3.3 Stereoisomers of Isoleucine Ile has 2 chiral carbons, 4 possible stereoisomers

17 Prentice Hall c2002Chapter 317 Proline has a nitrogen in the aliphatic ring system Proline (Pro, P) - has a three carbon side chain bonded to the  -amino nitrogen The heterocyclic pyrrolidine ring restricts the geometry of polypeptides

18 Prentice Hall c2002Chapter 318 B. Aromatic R Groups Side chains have aromatic groups Phenylalanine (Phe, F) - benzene ring Tyrosine (Tyr, Y) - phenol ring Tryptophan (Trp, W) - bicyclic indole group

19 Prentice Hall c2002Chapter 319 Aromatic amino acid structures

20 Prentice Hall c2002Chapter 320 C. Sulfur-Containing R Groups Methionine (Met, M) - (-CH 2 CH 2 SCH 3 ) Cysteine (Cys, C) - (-CH 2 SH) Two cysteine side chains can be cross-linked by forming a disulfide bridge (-CH 2 -S-S-CH 2 -) Disulfide bridges may stabilize the three- dimensional structures of proteins

21 Prentice Hall c2002Chapter 321 Methionine and cysteine

22 Prentice Hall c2002Chapter 322 Fig 3.4 Formation of cystine

23 Prentice Hall c2002Chapter 323 D. Side Chains with Alcohol Groups Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains

24 Prentice Hall c2002Chapter 324 E. Basic R Groups Histidine (His, R) - imidazole Lysine (Lys, K) - alkylamino group Arginine (Arg, R) - guanidino group Side chains are nitrogenous bases which are substantially positively charged at pH 7

25 Prentice Hall c2002Chapter 325 Structures of histidine, lysine and arginine

26 Prentice Hall c2002Chapter 326 F. Acidic R Groups and Amide Derivatives Aspartate (Asp, D) and Glutamate (Glu, E) are dicarboxylic acids, and are negatively charged at pH 7 Asparagine (Asn, N) and Glutamine (Gln, Q) are uncharged but highly polar

27 Prentice Hall c2002Chapter 327 Structures of aspartate, glutamate, asparagine and glutamine

28 Prentice Hall c2002Chapter 328 G. The Hydrophobicity of Amino Acid Side Chains Hydropathy: the relative hydrophobicity of each amino acid The larger the hydropathy, the greater the tendency of an amino acid to prefer a hydrophobic environment Hydropathy affects protein folding: hydrophobic side chains tend to be in the interior hydrophilic residues tend to be on the surface

29 Prentice Hall c2002Chapter 329 Table 3.1 Hydropathy scale for amino acid residues (Free-energy change for transfer of an amino acid from interior of a lipid bilayer to water) Free-energy change for transfer (kjmol -1 ) Amino acid

30 Prentice Hall c2002Chapter Other Amino Acids and Amino Acid Derivatives Over 200 different amino acids are found in nature Most are precursors to common amino acids or chemically modified derivatives Some amino acids are chemically modified after incorporation into a polypeptide

31 Prentice Hall c2002Chapter 331 Fig 3.5 Compounds derived from common amino acids

32 Prentice Hall c2002Chapter 332 Selenocysteine Selenocysteine is incorporated into a few proteins Constitutes the 21 st amino acid

33 Prentice Hall c2002Chapter Ionization of Amino Acids Ionizable groups in amino acids: (1)  -carboxyl, (2)  -amino, (3) some side chains Each ionizable group has a specific pK a AHB + H + For a solution pH below the pK a, the protonated form predominates (AH) For a solution pH above the pK a, the unprotonated form predominates (B)

34 Prentice Hall c2002Chapter 334 Fig 3.6 Titration curve for alanine Titration curves are used to determine pK a values pK 1 = 2.4 pK 2 = 9.9 pI Ala = isoelectric point

35 Prentice Hall c2002Chapter 335 Fig 3.7 Ionization of Histidine (a) Titration curve of histidine pK 1 = 1.8 pK 2 = 6.0 pK 3 = 9.3

36 Prentice Hall c2002Chapter 336 Fig 3.7 (b) Deprotonation of imidazolium ring

37 Prentice Hall c2002Chapter 337 Table 3.2 pK a values of amino acid ionizable groups

38 Prentice Hall c2002Chapter 338 Henderson-Hasselbach equation: calculating group ionizations [proton acceptor] pH = pK a + log [proton donor]

39 Prentice Hall c2002Chapter 339 Fig 3.8 (a) Ionization of the protonated  -carboxyl of glutamate

40 Prentice Hall c2002Chapter 340 Fig 3.8 (b) Deprotonation of the guanidinium group of Arg

41 Prentice Hall c2002Chapter Peptide Bonds Link Amino Acids in Proteins Peptide bond - linkage between amino acids is a secondary amide bond Formed by condensation of the  -carboxyl of one amino acid with the  -amino of another amino acid (loss of H 2 O molecule) Primary structure - linear sequence of amino acids in a polypeptide or protein

42 Prentice Hall c2002Chapter 342 Fig 3.9 Peptide bond between two amino acids

43 Prentice Hall c2002Chapter 343 Polypeptide chain nomenclature Amino acid “residues” compose peptide chains Peptide chains are numbered from the N (amino) terminus to the C (carboxyl) terminus Example: (N) Gly-Arg-Phe-Ala-Lys (C) (or GRFAK) Formation of peptide bonds eliminates the ionizable  -carboxyl and  -amino groups of the free amino acids

44 Prentice Hall c2002Chapter 344 Fig 3.10 Aspartame, an artificial sweetener Aspartame is a dipeptide methyl ester (aspartylphenylalanine methyl ester) About 200 times sweeter than table sugar Used in diet drinks

45 Prentice Hall c2002Chapter Protein Purification Techniques Common types of column chromatography: Ion-exchange chromatography - separation based upon the overall charge of molecules Gel-filtration chromatography - separation based upon molecular size Affinity chromatography - separation by specific binding interactions between column matrix and target proteins

46 Prentice Hall c2002Chapter 346 Fig 3.11 Column Chromatography (a) Separation of a protein mixture (b) Detection of eluting protein peaks

47 Prentice Hall c2002Chapter 347 Electrophoresis Polyacrylamide gel electrophoresis (PAGE) Separates molecules on a polyacrylamide gel matrix when an electric field is applied SDS-PAGE. Sodium dodecyl sulfate (SDS) coats proteins with negative charges. Coated polypeptide chains then separate by molecular mass (method to determine molecular weight)

48 Prentice Hall c2002Chapter 348 Fig 3.12 (a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run

49 Prentice Hall c2002Chapter 349

50 Prentice Hall c2002Chapter 350

51 Prentice Hall c2002Chapter 351

52 Prentice Hall c2002Chapter 352 Fig. 3.14

53 Prentice Hall c2002Chapter 353

54 Prentice Hall c2002Chapter 354

55 Prentice Hall c2002Chapter 355

56 Prentice Hall c2002Chapter Amino Acid Composition of Proteins Amino acid analysis - determination of the amino acid composition of a protein Peptide bonds are cleaved by acid hydrolysis (6M HCl, 110 o, hours) Amino acids are separated chromatographically and quantitated Phenylisothiocyanate (PITC) used to derivatize the amino acids prior to HPLC analysis

57 Prentice Hall c2002Chapter 357 Fig 3.15 Acid-catalyzed hydrolysis of a peptide

58 Prentice Hall c2002Chapter 358 Fig 3.16 Amino acid treated with PITC

59 Prentice Hall c2002Chapter 359 Fig 3.17 Chromatogram from HPLC- separated PTC-amino acids

60 Prentice Hall c2002Chapter Determining the Sequence of Amino Acids Edman degradation procedure - Determining one residue at a time from the N-terminus (1) Treat peptide with PITC which reacts with the N-terminus to form a PTC-peptide (2) Treat with trifluoroacetic acid (TFA) to selectively cleave the N-terminal peptide bond (3) Separate N-terminal derivative from peptide (4) Convert derivative to PTH-amino acid

61 Prentice Hall c2002Chapter 361 Fig 3.18 Edman degradation procedure Phenylisothiocyanate (Edman reagent) pH = 9.0 Phenylthiocarbamoyl-peptide F 3 CCOOH

62 Prentice Hall c2002Chapter 362 Edman degradation procedure (cont) Aqueous acid Polypeptide chain with n-1 amino acids Returned to alkaline conditions for reaction with additional phenylisothiocyanate in the next cycle of Edman degradation

63 Prentice Hall c2002Chapter 363 Cleaving and blocking disulfide bonds Disulfide bonds in proteins must be cleaved: (1) To permit isolation of the PTH-cysteine during the Edman procedure (2) To separate peptide chains Treatment with thiol compounds reduces the (R-S-S-R) cystine bond to two cysteine (R-SH) residues

64 Prentice Hall c2002Chapter 364 Fig 3.19 Cleaving, blocking disulfide bonds

65 Prentice Hall c2002Chapter Protein Sequencing Strategies Proteins may be too large to be sequenced completely by the Edman method Proteases (enzymes cleaving peptide bonds) and chemical agents are used to selectively cleave the protein into smaller fragments Cyanogen bromide (BrCN) cleaves polypeptides at the C-terminus of Met residues

66 Prentice Hall c2002Chapter 366 Fig 3.20 Protein cleavage by BrCN

67 Prentice Hall c2002Chapter 367 Protease enzymes cleave specific peptide bonds Chymotrypsin - carbonyl side of aromatic or bulky noncharged aliphatic residues (e.g. Phe, Tyr, Trp, Leu) Trypsin - carbonyl side, basic residues (Lys,Arg). Staphylococcus aureus V8 protease - carbonyl side of negatively charged residues (Glu, Asp). NOTE: in 50mM ammonium bicarbonate cleaves only at Glu.

68 Prentice Hall c2002Chapter 368 Fig 3.21 Cleavage, sequencing an oligopeptide

69 Prentice Hall c2002Chapter 369 Fig 3.20 Sequences of DNA and protein Protein amino acid sequences can be deduced from the sequence of nucleotides in the corresponding gene A sequence of three nucleotides specifies one amino acid (A,C,G,T are DNA residues )

70 Prentice Hall c2002Chapter Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships Closely related species contain proteins with very similar amino acid sequences Differences reflect evolutionary change from a common ancestral protein sequence Cytochrome c protein sequences from various species can be aligned to show their similarities Phylogenetic tree shows evolutionary differences in amino acid sequences

71 Prentice Hall c2002Chapter 371 Fig. 3.23

72 Prentice Hall c2002Chapter 372 Fig 3.24 Phylogenetic tree for cytochrome c

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