1. Chapter 3: Amino Acids, Peptides, and Proteins
Dr. Clower
Chem 4202

2. Outline (part I) Sections 3.1 and 3.2 Amino Acids
Chemical structure
Acid-base properties
Stereochemistry
Non-standard amino acids
Formation of Peptide Bonds

3. Amino Acids The building blocks of proteins
Also used as single molecules in biochemical pathways
20 standard amino acids (a-amino acids)
Two functional groups:
carboxylic acid group
amino group on the alpha () carbon
Have different side groups (R)
Properties dictate behavior of AAs
R side chain |
H2N— C —COOH | H

4. Zwitterions
Both the –NH2 and the –COOH groups in an amino acid undergo ionization in water.
At physiological pH (7.4), a zwitterion forms
Both + and – charges
Overall neutral
Amphoteric
Amino group is protonated
Carboxyl group is deprotonated
Soluble in polar solvents due to ionic character
Structure of R also influence solubility

5. Classification of Amino Acids
Classify by structure of R
Nonpolar
Polar
Aromatic
Acidic
Basic

6. Nonpolar Amino Acids
Hydrophobic, neutral, aliphatic

7. Polar Amino Acids
Hydrophilic, neutral, typically H-bond

8. Disulfide Bonds
Formed from oxidation of cysteine residues

9. Aromatic Amino Acids
Bulky, neutral, polarity depend on R

10. Acidic and Basic Amino Acids
R group = carboxylic acid
Donates H+
Negatively charged
Basic
R group = amine
Accepts H+
Positively charged
His ionizes at pH 6.0

11. Acid-base Properties Remember H3PO4 (multiple pKa’s)
AAs also have multiple pKa’s due to multiple ionizable groups
pK1 ~ 2.2
(protonated below 2.2)
pK2 ~ 9.4
(NH3+ below 9.4)
pKR
(when applicable)

12. Table 3-1 Note 3-letter and 1-letter abbreviations
Amino acid organization chart

13. pH and Ionization Consider glycine:
Note that the uncharged species never forms

14. Titration of Glycine pK1 pK2 First equivalence point Animation
[cation] = [zwitterion]
pK2
[zwitterion] = [anion]
First equivalence point
Zwitterion
Molecule has no net charge
pH = pI (Isoelectric point)
pI = average of pKa’s = ½ (pK1 + pK2)
pIglycine = ½ ( ) = 5.97
Animation

15. pI of Lysine
For AAs with 3 pKa’s, pI = average of two relevant pKa values
Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
Which species is the isoelectric form?
So, pI = ½ (pK2 + pKR)
= ½ ( ) = 9.74
Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)

16. Learning Check
Would the following ions of serine exist at a pH above, below, or at pI?

17. Stereochemistry of AAs
All amino acids (except glycine) are optically active
Fischer projections:

18. D and L Configurations d = dextrorotatory l = levorotatory
D, L = relative to glyceraldehyde

19. Importance of Stereochemistry
All AA’s found in proteins are L geometry
S enantiomer for all except cysteine
D-AA’s are found in bacteria
Geometry of proteins affects reactivity (e.g binding of substrates in enzymes)
Thalidomide

20. Non-standard Amino Acids
AA derivatives
Modification of AA after protein synthesized
Terminal residues or R groups
Addition of small alkyl group, hydroxyl, etc.
D-AAs
Bacteria

21. CHEM 2412 Review
Carboxylic acid + amine = ?
Structure of amino acid

22. The Peptide Bond Chain of amino acids = peptide or protein
Amino acid residues connected by peptide bonds
Residue = AA – H2O

23. The Peptide Bond
Non-basic and non-acidic in pH 2-12 range due to delocalization of N lone pair
Amide linkage is planar, NH and CO are anti
Rigid
restricted rotation

24. Polypeptides Linear polymers (no branches)
AA monomers linked head to tail
Terminal residues:
Free amino group (N-terminus)
Draw on left
Free carboxylate group (C-terminus)
Draw on right
pKa values of AAs in polypeptides differ slightly from pKa values of free AAs

25. Naming Peptides Name from the free amine (NH3+)
Use -yl endings for the names of the amino acids
The last amino acid with the free carboxyl group (COO-) uses its amino acid name
(GA)

26. Amino Acid Ambiguity Glutamate (Glu/E) vs. Glutamine (Gln/Q)
Aspartate (Asp/D) vs. Asparagine (Asn/N)
Converted via hydrolysis
Use generic abbreviations for either
Glx/Z
Asx/B
X = undetermined or nonstandard AA

27. Learning Check
Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.

28. Learning Check
Draw the structural formula of each of the following peptides.
A. Methionylaspartic acid
B. Alanyltryptophan
C. Methionylglutaminyllysine
D. Histidylglycylglutamylalanine

29. Outline (part II) Sections 3.3 and 3.4 Separation and purification
Protein sequencing
Analysis of primary structure

30. Protein size In general, proteins contain > 40 residues
Minimum needed to fold into tertiary structure
Usually residues
Percent of each AA varies
Proteins separated based on differences in size and composition
Proteins must be pure to analyze, determine structure/function

31. Factors to control pH Presence of enzymes Temperature Thiol groups
Keep pH stable to avoid denaturation or chemical degradation
Presence of enzymes
May affect structure (e.g. proteases/peptidase)
Temperature
Control denaturation (0-4°C)
Control activity of enzymes
Thiol groups
Reactive
Add protecting group to prevent formation of new disulfide bonds
Exposure to air, water
Denature or oxidize
Store under N2 or Ar
Keep concentration high

32. General Separation Procedure
Detect/quantitate protein (assay)
Determine a source (tissue)
Extract protein
Suspend cell source in buffer
Homogenize
Break into fine pieces
Cells disrupted
Soluble contents mix with buffer
Centrifuge to separate soluble and insoluble
Separate protein of interest
Based on solubility, size, charge, or binding ability

33. Solubility Selectively precipitate protein Manipulate
Concentration of salts
Solvent pH
Temperature

34. Concentration of salts
Adding small amount of salt increases [Protein]
Salt shields proteins from each other, less precipitation from aggregation
Salting-in
Salting out
Continue to increase [salt] decreases [protein]
Different proteins salt out at different [salt]

35. Other Solubility Methods
Solvent
Similar theory to salting-out
Add organic solvent (acetone, ethanol) to interact with water
Decrease solvating power pH
Proteins are least soluble at pI
Isoelectric precipitation
Temperature
Solubility is temperature dependent

36. Chromatography Mobile phase Stationary phase
Mixture dissolved in liquid or solid
Stationary phase
Porous solid matrix
Components of mixture pass through the column at different rates based on properties

37. Types of Chromatography
Paper
Stationary phase = filter paper
Same theory as thin layer chromatography (TLC)
Components separate based on polarity
High-performance liquid (HPLC)
Stationary phase = small uniform particles, large surface area
Adapt to separate based on polarity, size, etc.
Hydrophobic Interaction
Hydrophobic groups on matrix
Attract hydrophobic portions of protein

38. Types of Chromatography
Ion-exchange
Stationary phase = chemically modified to include charged groups
Separate based on net charge of proteins
Anion exchangers
Cation groups (protonated amines) bind anions
Cation exchangers
Anion groups (carboxylates) bind cations

39. Types of Chromatography
Gel-filtration
Size/molecular exclusion chromatography
Stationary phase = gels with pores of particular size
Molecules separate based on size
Small molecules caught in pores
Large molecules pass through

40. Types of Chromatography
Affinity
Matrix chemically altered to include a molecule designed to bind a particular protein
Other proteins pass through

41. UV-Vis Spectroscopy
Absorbance used to monitor protein concentrations of each fraction
l = 280 nm
Absorbance of aromatic side groups

42. Electrophoresis Migration of ions in an electric field
Electrophoretic mobility (rate of movement) function of charge, size, voltage, pH
The positively charged proteins move towards the negative electrode (cathode)
The negatively charged proteins move toward the positive electrode (anode)
A protein at its pI (neutral) will not migrate in either direction
Variety of supports (gel, paper, starch, solutions)

43. Protein Sequencing Determination of primary structure
Need to know to determine 3D structure
Gives insight into protein function
Approach:
Denature protein
Break protein into small segments
Determine sequences of segments
Animation

44. End group analysis Identify number of terminal AAs
Number of chains/subunits
Identify specific AA
Dansyl chloride/dabsyl chloride
Sanger method (FDNB)
Edman degradation (PITC)
Bovine insulin

45. Dansyl chloride Reacts with primary amines
N-terminus
Yields dansylated polypeptides
Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA
Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)
Useful method when protein fragmented into shorter polypeptides

46. Dabsyl chloride and FDNB
Same result as dansyl chloride
Dabsyl chloride
1-Fluoro-2,4-dinitrobenzene (FDNB)
Sanger method

47. Edman degradation Phenylisothiocyanate (PITC)
Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC)
Treat with TFAA (solvent/catalyst) to cleave N-terminal residue
Does not hydrolyze other AAs
Treatment with dilute acid makes more stable organic compound
Identify using NMR, HPLC, etc.
Sequenator (entire process for proteins < 100 residues)

48. Fragmenting Proteins
Formation of smaller segments to assist with sequencing
Process:
Cleave protein into specific fragments
Chemically or enzymatically
Break disulfide bonds
Purify fragments
Sequence fragments
Determine order of fragments and disulfide bonds

49. Cleaving Disulfide Bonds
Oxidize with performic acid
Cys residues form cysteic acid
Acid can oxidize other residues, so not ideal

50. Cleaving Disulfide Bonds
Reduce by mercaptans (-SH)
2-Mercaptoethanol
HSCH2CH2OH
Dithiothreitol (DTT)
HSCH2CH(OH)CH(OH)CH2SH
Reform cysteine residues
Oxidize thiol groups with iodoacetete (ICH2CO2-) to prevent reformation of disulfide bonds

51. Hydrolysis Cleaves all peptide bonds Achieved by After cleavage:
Enzyme
Acid
Base
After cleavage:
Identify using chromatography
Quantify using absorbance or fluorescence
Disadvantages
Doesn’t give exact sequence, only AAs present
Acid and base can degrade/modify other residues
Enzymes (which are proteins) can also cleave and affect results

52. Enzymatic and Chemical Cleavage
Enzymes used to break protein into smaller peptides
Endopeptidases
Catalyze hydrolysis of internal peptide bonds
Chemical
Chemical reagents used to break up polypeptides
Cyanogen bromide (BrCN)

53. An example

54. Fundamentals of Protein Structure

55. Our life is maintained by molecular network systems
Molecular network system in a cell
(From ExPASy Biochemical Pathways;

56. Proteins play key roles in a living system
Three examples of protein functions
Catalysis: Almost all chemical reactions in a living cell are catalyzed by protein enzymes.
Transport: Some proteins transports various substances, such as oxygen, ions, and so on.
Information transfer: For example, hormones.
Alcohol dehydrogenase oxidizes alcohols to aldehydes or ketones
Haemoglobin carries oxygen
Insulin controls the amount of sugar in the blood

57. Amino acid: Basic unit of protein
COO-
NH3+ C R H
Different side chains, R, determin the properties of 20 amino acids.
Amino group
Carboxylic acid group
An amino acid

58. White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
20 Amino acids
Glycine (G)
Alanine (A)
Valine (V)
Isoleucine (I)
Leucine (L)
Proline (P)
Methionine (M)
Phenylalanine (F)
Tryptophan (W)
Asparagine (N)
Glutamine (Q)
Serine (S)
Threonine (T)
Tyrosine (Y)
Cysteine (C)
Asparatic acid (D)
Glutamic acid (E)
Lysine (K)
Arginine (R)
Histidine (H)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

59. Proteins are linear polymers of amino acids
NH3＋ C
COOー ＋
NH3＋ C
COOー ＋ H H
A carboxylic acid condenses with an amino group with the release of a water
H2O
H2O R1 R2 R3
NH3＋ C CO NH C CO NH C CO H
Peptide bond H
Peptide bond H
The amino acid sequence is called as primary structure F T D A G S K A N G S

60. Amino acid sequence is encoded by DNA base sequence in a gene
DNA molecule
DNA base sequence ・ C G A T ＝

61. Amino acid sequence is encoded by DNA base sequence in a gene
Second letter T C A G
First letter
TTT
Phe
TCT
Ser
TAT
Tyr
TGT
Cys
Third letter
TTC
TCC
TAC
TGC
TTA
Leu
TCA
TAA
Stop
TGA
TTG
TCG
TAG
TGG
Trp
CTT
CCT
Pro
CAT
His
CGT
Arg
CTC
CCC
CAC
CGC
CTA
CCA
CAA
Gln
CGA
CTG
CCG
CAG
CGG
ATT
Ile
ACT
Thr
AAT
Asn
AGT
ATC
ACC
AAC
AGC
ATA
ACA
AAA
Lys
AGA
ATG
Met
ACG
AAG
AGG
GTT
Val
GCT
Ala
GAT
Asp
GGT
Gly
GTC
GCC
GAC
GGC
GTA
GCA
GAA
Glu
GGA
GTG
GCG
GAG
GGG

62. Gene is protein’s blueprint, genome is life’s blueprint
DNA
Genome
Gene
Gene
Protein
Protein

63. Gene is protein’s blueprint, genome is life’s blueprint
Glycolysis network
Genome
Gene
Protein

64. In 2003, Human genome sequence was deciphered!
Genome is the complete set of genes of a living thing.
In 2003, the human genome sequencing was completed.
The human genome contains about 3 billion base pairs.
The number of genes is estimated to be between 20,000 to 25,000.
The difference between the genome of human and that of chimpanzee is only 1.23%!
3 billion base pair => 6 G letters
&
1 letter => 1 byte
The whole genome can be recorded in just 10 CD-ROMs!

65. Each Protein has a unique structure
Amino acid sequence
NLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG
Folding!

66. Basic structural units of proteins: Secondary structure
α-helix
β-sheet
Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.

67. Three-dimensional structure of proteins
Tertiary structure
Quaternary structure

68. Hierarchical nature of protein structure
Primary structure (Amino acid sequence) ↓
Secondary structure （α-helix, β-sheet）
Tertiary structure （Three-dimensional structure formed by assembly of secondary structures）
Quaternary structure （Structure formed by more than one polypeptide chains）

69. Close relationship between protein structure and its function
Example of enzyme reaction
Hormone receptor
Antibody
substrates A
enzyme
enzyme B
Matching the shape to A
Digestion of A!
enzyme A
Binding to A

70. Protein structure prediction has remained elusive over half a century
“Can we predict a protein structure from its amino acid sequence?”
Now, impossible!

71. Summary Proteins are key players in our living systems.
Proteins are polymers consisting of 20 kinds of amino acids.
Each protein folds into a unique three-dimensional structure defined by its amino acid sequence.
Protein structure has a hierarchical nature.
Protein structure is closely related to its function.
Protein structure prediction is a grand challenge of computational biology.