1. Chapter 3: Amino Acids, Peptides, and Proteins
Dr. Rajabi

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)( amino acid=Selenocysteine)
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?

18.  Histidine
A good buffer at
~ pH 6.
pI =

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

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

22. 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

23. 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

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

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

26. 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

27. 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

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

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

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

31. Protein structure: Primary structure:
There are four levels of protein structure (primary, secondary, tertiary and quaternary)
Primary structure:
  The primary structure of a protein is its unique sequence of amino acids.
Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids.
The precise primary structure of a protein is determined by inherited genetic information.
At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus).

32. 2- Secondary structure:
Results from hydrogen bond formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:
α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one
β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).

35. I →I+4

36. Hydrogen bonding in α-helix: In the α-helix CO of the one amino acid residue forms H-bond with NH of the forth one.
Supersecondary structure or Motifs :
occurs by combining secondary structure.
The combination may be: α-helix- turn- α-helix- turn…..etc
Or: β-sheet -turn- β-sheet-turn………etc
Or: α-helix- turn- β-sheet-turn- α-helix
Turn (or bend): is short segment of polypeptides (3-4 amino acids) that connects successive secondary structures.
e.g. β-turn: is small polypeptide that connects successive strands of β-sheets.

37. Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure.

38. Quaternary structure: results from the aggregation (combination) of two or more polypeptide subunits held together by non-covalent interaction like H-bonds, ionic or hydrophobic interactions.
Examples on protein having quaternary structure:
Collagen is a fibrous protein of three polypeptides (trimeric) that are supercoiled like a rope.
This provides the structural strength for their role in connective tissue.
Hemoglobin is a globular protein with four polypeptide chains (tetrameric)
Insulin : two polypeptide chains (dimeric)

40. Summary of Protein Structures
Copyright © by Pearson Education, Inc Publishing as Benjamin Cummings

41. 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

42. 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

43. 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

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

45. 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]

46. 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

47. 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

48. 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

49. 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

50. 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

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

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

53. 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)

54. 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

55. 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

56. 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

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

58. 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)

59. 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

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

61. 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

62. 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

63. 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)

64. An example

65. Another example
A protein is cleaved with cyanogen bromide to yield the following sequences:
Arg-Ala-Tyr-Gly-Asn
Leu-Phe-Met
Asp-Met
The same protein is cleaved with chymotrypsin to yield the following sequences:
Met-Arg-Ala-Tyr
Asp-Met-Leu-Phe
Gly-Asn
What is the sequence of the protein?

66. Suggested Problems, Chapter 3
1-5, 7, 10-13, 15, 18

67. 2- Secondary structure:
Results from hydrogen bond formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:
α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one
β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).