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

2. 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)
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3. 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)
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4. 3.1 General Structure of Amino Acids
Twenty common a-amino acids have carboxyl and amino groups bonded to the a-carbon atom
A hydrogen atom and a side chain (R) are also attached to the a-carbon atom
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5. Zwitterionic form of amino acids
Under normal cellular conditions amino acids are zwitterions (dipolar ions):
Amino group = -NH3+
Carboxyl group = -COO-
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6. Fig 3.1 Two representations of an amino acid at neutral pH
(a) Structure
(b) Ball-and stick model
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7. 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
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8. Stereochemistry of amino acids
19 of the 20 common amino acids have a chiral a-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)
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9. Fig 3.2 Mirror-image pairs of amino acids
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10. 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 -CH2OH)
Priorities (low to high): -H, -CH3, -C6H5, -CH2OH, -CHO, -COOH, -NH2, -NHR, -OH, -OR, -SH
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11. 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.
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12. Assignment of RS configuration
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13. 3.2 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
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14. A. Aliphatic R Groups
Glycine (Gly, G) - the a-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 a-C and N
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15. Four aliphatic amino acid structures
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16. Fig 3.3 Stereoisomers of Isoleucine
Ile has 2 chiral carbons, 4 possible stereoisomers
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17. Proline has a nitrogen in the aliphatic ring system
Proline (Pro, P) - has a three carbon side chain bonded to the a-amino nitrogen
The heterocyclic pyrrolidine ring restricts the geometry of polypeptides
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18. 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
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19. Aromatic amino acid structures
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20. C. Sulfur-Containing R Groups
Methionine (Met, M) - (-CH2CH2SCH3)
Cysteine (Cys, C) - (-CH2SH)
Two cysteine side chains can be cross-linked by forming a disulfide bridge (-CH2-S-S-CH2-)
Disulfide bridges may stabilize the three-dimensional structures of proteins
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21. Methionine and cysteine
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22. Fig 3.4 Formation of cystine
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23. D. Side Chains with Alcohol Groups
Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains
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24. 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
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25. Structures of histidine, lysine and arginine
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26. 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
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27. Structures of aspartate, glutamate, asparagine and glutamine
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28. 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
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29. Table 3.1 Hydropathy scale for amino acid residues
Free-energy change for transfer (kjmol-1)
Hydropathy scale for amino acid residues
(Free-energy change for transfer of an amino acid from interior of a lipid bilayer to water)
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30. 3.3 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
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31. Fig 3.5 Compounds derived from common amino acids
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32. Selenocysteine is incorporated into a few proteins
Constitutes the 21st amino acid
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33. 3.4 Ionization of Amino Acids
Ionizable groups in amino acids: (1) a-carboxyl, (2) a-amino, (3) some side chains
Each ionizable group has a specific pKa
AH B + H+
For a solution pH below the pKa, the protonated form predominates (AH)
For a solution pH above the pKa, the unprotonated form predominates (B)
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34. Fig 3.6 Titration curve for alanine
Titration curves are used to determine pKa values
pK1 = 2.4
pK2 = 9.9
pIAla = isoelectric point
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35. Fig 3.7 Ionization of Histidine
(a) Titration curve of histidine
pK1 = 1.8 pK2 = 6.0 pK3 = 9.3
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36. Fig 3.7 (b) Deprotonation of imidazolium ring
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37. Table 3.2 pKa values of amino acid ionizable groups
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38. Henderson-Hasselbach equation: calculating group ionizations
[proton acceptor]
pH = pKa + log
[proton donor]
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39. Fig 3.8 (a) Ionization of the protonated g-carboxyl of glutamate
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40. Fig 3.8 (b) Deprotonation of the guanidinium group of Arg
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41. 3.5 Peptide Bonds Link Amino Acids in Proteins
Peptide bond - linkage between amino acids is a secondary amide bond
Formed by condensation of the a-carboxyl of one amino acid with the a-amino of another amino acid (loss of H2O molecule)
Primary structure - linear sequence of amino acids in a polypeptide or protein
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42. Fig 3.9 Peptide bond between two amino acids
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43. 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 a-carboxyl and a-amino groups of the free amino acids
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44. 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
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45. 3.6 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
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46. Fig 3.11 Column Chromatography
(a) Separation of a protein mixture
(b) Detection of eluting protein peaks
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47. 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)
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48. Fig 3. 12 (a) SDS-PAGE Electrophoresis
Fig 3.12 (a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run
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50. Prentice Hall c2002
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51. Prentice Hall c2002
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52. Fig. 3.14
Figure 3.14
MALDI–TOF mass spectrometry. (a) A burst of light releases proteins from the matrix. (b) Charged proteins are directed toward the detector by an electric field. (c) The time of arrival at the detector depends on the mass and the charge of the protein.
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53. Figure 3.14
MALDI–TOF mass spectrometry. (a) A burst of light releases proteins from the matrix. (b) Charged proteins are directed toward the detector by an electric field. (c) The time of arrival at the detector depends on the mass and the charge of the protein.
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54. Figure 3.14
MALDI–TOF mass spectrometry. (a) A burst of light releases proteins from the matrix. (b) Charged proteins are directed toward the detector by an electric field. (c) The time of arrival at the detector depends on the mass and the charge of the protein.
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56. 3.7 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, 110o, hours)
Amino acids are separated chromatographically and quantitated
Phenylisothiocyanate (PITC) used to derivatize the amino acids prior to HPLC analysis
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57. Fig 3.15 Acid-catalyzed hydrolysis of a peptide
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58. Fig 3.16 Amino acid treated with PITC
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59. Fig 3.17 Chromatogram from HPLC-separated PTC-amino acids
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60. 3.8 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
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61. Fig 3.18 Edman degradation procedure
Phenylisothiocyanate (Edman reagent)
pH = 9.0
Phenylthiocarbamoyl-peptide
F3CCOOH
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62. Edman degradation procedure (cont)
Polypeptide chain with n-1 amino acids
Aqueous acid
Returned to alkaline conditions for reaction with additional phenylisothiocyanate in the next cycle of Edman degradation
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63. 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
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64. Fig 3.19 Cleaving, blocking disulfide bonds
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65. 3.9 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
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66. Fig 3.20 Protein cleavage by BrCN
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67. 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.
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68. Fig 3.21 Cleavage, sequencing an oligopeptide
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69. 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 )
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70. 3.10 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
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71. Fig. 3.23
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72. Fig 3.24 Phylogenetic tree for cytochrome c Prentice Hall c2002
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