Principles of Biochemistry Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and the Primary Structures of Proteins Prentice Hall c2002 Chapter 3 Copyright © 2006 Pearson Prentice Hall, Inc.

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) Prentice Hall c2002 Chapter 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) Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Zwitterionic form of amino acids Under normal cellular conditions amino acids are zwitterions (dipolar ions): Amino group = -NH3+ Carboxyl group = -COO- Prentice Hall c2002 Chapter 3

Fig 3.1 Two representations of an amino acid at neutral pH (a) Structure (b) Ball-and stick model Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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) Prentice Hall c2002 Chapter 3

Fig 3.2 Mirror-image pairs of amino acids Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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. Prentice Hall c2002 Chapter 3

Assignment of RS configuration Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Four aliphatic amino acid structures Prentice Hall c2002 Chapter 3

Fig 3.3 Stereoisomers of Isoleucine Ile has 2 chiral carbons, 4 possible stereoisomers Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Aromatic amino acid structures Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Methionine and cysteine Prentice Hall c2002 Chapter 3

Fig 3.4 Formation of cystine Prentice Hall c2002 Chapter 3

D. Side Chains with Alcohol Groups Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Structures of histidine, lysine and arginine Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Structures of aspartate, glutamate, asparagine and glutamine Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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) Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.5 Compounds derived from common amino acids Prentice Hall c2002 Chapter 3

Selenocysteine is incorporated into a few proteins Constitutes the 21st amino acid Prentice Hall c2002 Chapter 3

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) Prentice Hall c2002 Chapter 3

Fig 3.6 Titration curve for alanine Titration curves are used to determine pKa values pK1 = 2.4 pK2 = 9.9 pIAla = isoelectric point Prentice Hall c2002 Chapter 3

Fig 3.7 Ionization of Histidine (a) Titration curve of histidine pK1 = 1.8 pK2 = 6.0 pK3 = 9.3 Prentice Hall c2002 Chapter 3

Fig 3.7 (b) Deprotonation of imidazolium ring Prentice Hall c2002 Chapter 3

Table 3.2 pKa values of amino acid ionizable groups Prentice Hall c2002 Chapter 3

Henderson-Hasselbach equation: calculating group ionizations [proton acceptor] pH = pKa + log [proton donor] Prentice Hall c2002 Chapter 3

Fig 3.8 (a) Ionization of the protonated g-carboxyl of glutamate Prentice Hall c2002 Chapter 3

Fig 3.8 (b) Deprotonation of the guanidinium group of Arg Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.9 Peptide bond between two amino acids Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.11 Column Chromatography (a) Separation of a protein mixture (b) Detection of eluting protein peaks Prentice Hall c2002 Chapter 3

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) Prentice Hall c2002 Chapter 3

Fig 3. 12 (a) SDS-PAGE Electrophoresis Fig 3.12 (a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run Prentice Hall c2002 Chapter 3

Prentice Hall c2002 Chapter 3

Prentice Hall c2002 Chapter 3

Prentice Hall c2002 Chapter 3

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. Prentice Hall c2002 Chapter 3

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. Prentice Hall c2002 Chapter 3

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. Prentice Hall c2002 Chapter 3

Prentice Hall c2002 Chapter 3

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, 16-72 hours) Amino acids are separated chromatographically and quantitated Phenylisothiocyanate (PITC) used to derivatize the amino acids prior to HPLC analysis Prentice Hall c2002 Chapter 3

Fig 3.15 Acid-catalyzed hydrolysis of a peptide Prentice Hall c2002 Chapter 3

Fig 3.16 Amino acid treated with PITC Prentice Hall c2002 Chapter 3

Fig 3.17 Chromatogram from HPLC-separated PTC-amino acids Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.18 Edman degradation procedure Phenylisothiocyanate (Edman reagent) pH = 9.0 Phenylthiocarbamoyl-peptide F3CCOOH Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.19 Cleaving, blocking disulfide bonds Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig 3.20 Protein cleavage by BrCN Prentice Hall c2002 Chapter 3

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. Prentice Hall c2002 Chapter 3

Fig 3.21 Cleavage, sequencing an oligopeptide Prentice Hall c2002 Chapter 3

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 ) Prentice Hall c2002 Chapter 3

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 Prentice Hall c2002 Chapter 3

Fig. 3.23 Prentice Hall c2002 Chapter 3

Fig 3.24 Phylogenetic tree for cytochrome c Prentice Hall c2002 Chapter 3