1. CHAPTER 2 Amino Acids, Peptides, Proteins
Structure and naming of amino acids
Structure and properties of peptides
Ionization behavior of amino acids and peptides
Purification and assay methods
Peptide sequencing and chemical synthesis
Protein sequence analysis

2. Proteins: Main Agents of Biological Function
Catalysis:
enolase (in the glycolytic pathway)
DNA polymerase (in DNA replication)
Transport:
hemoglobin (transports O2 in the blood)
lactose permease (transports lactose across the cell membrane)
Structure:
collagen (connective tissue)
keratin (hair, nails, feathers, horns)
Motion:
myosin (muscle tissue)
actin (muscle tissue, cell motility)

3. Amino Acids: Building Blocks of Protein
Proteins are heteropolymers of -amino acids
Amino acids have properties that are well suited to carry out a variety of biological functions:
Capacity to polymerize
Useful acid-base properties
Varied physical properties
Varied chemical functionality

4. Amino Acids: Atom Naming
Organic nomenclature: start from one end
Biochemical designation: start from
-carbon and go down the R-group

5. Most -Amino Acids are Chiral
The -carbon has always four substituents and is tetrahedral
All (except proline) have an acidic carboxyl group, a basic amino group, and an alpha hydrogen connected to the -carbon
Each amino acid has an unique fourth substituent R
In glycine, the fourth substituent is also hydrogen

6. Amino Acids: Classification
Common amino acids can be placed in five basic groups depending on their R substituents:
Nonpolar, aliphatic (7)
Aromatic (3)
Polar, uncharged (5)
Positively charged (3)
Negatively charged (2)

7. Aliphatic Amino Acids

8. Aromatic Amino Acids

9. Charged Amino Acids

10. Polar Amino Acids

11. Special Amino Acids

13. Uncommon Amino Acids in Proteins
Not incorporated by ribosomes
Arise by post-translational modifications of proteins
Reversible modifications, esp. phosphorylation is important in regulation and signaling

14. The Genetic Code is organized by Amino Acid Properties

15. Ionization
At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form
At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions
At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form.

16. Substituent effects on pKa Values
-carboxy group is much more acidic than in carboxylic acids
-amino group is slightly less basic than in amines

17. Amino Acids Can Act as Buffers
Amino acids with uncharged side-chains, such as glycine, have two pKa values:
The pKa of the -carboxyl group is 2.34
The pKa of the -amino group is 9.6
It can act as a buffer in two pH regimes.

18. Amino Acids Carry a Net Charge of Zero at a Specific pH
Zwitterions predominate at pH values between the pKa values of amino and carboxyl group
For amino acid without ionizable side chains, the Isoelectric Point (equivalence point, pI) is
At this point, the net charge is zero
AA is least soluble in water
AA does not migrate in electric field

19. Ionizable Side Chains Can Show Up in Titration Curves
Ionizable side chains can be also titrated
Titration curves are now more complex
pKa values are discernable if two pKa values are more than two pH units apart
Why is the side-chain pKa so much higher?

20. How to Calculate the pI When the Side-chain is Ionizable?
Identify species that carries a net zero charge
Identify pKa value that defines the acid strength of this zwitterion: (pK2)
Identify pKa value that defines the base strength of this zwitterion: (pKR)
Take the average of these two pKa values

21. Peptides and Peptide bonds
Peptide bond in a di-peptide
“Peptides” are small condensation products of amino acids
They are “small” compared to proteins (di, tri, tetra… oligo-)

22. Peptide Ends are Not the Same
Numbering starts from the amino terminus
AA AA AA AA AA5

23. The Three Letter Code Naming starts from the N-terminus
Sequence is written as:
Ala-Glu-Gly-Lys
Sometimes the one-letter code is used:
AEGK

24. Peptides: A Variety of Functions
Hormones and pheromones:
insulin (think sugar)
oxytocin (think childbirth)
sex-peptide (think fruit fly mating)
Neuropeptides
substance P (pain mediator)
Antibiotics:
polymyxin B (for Gram - bacteria)
bacitracin (for Gram + bacteria)
Protection, e.g. toxins
amanitin (mushrooms)
conotoxin (cone snails)
chlorotoxin (scorpions)

25. Proteins are:
Polypeptides (covalently linked -amino acids) + possibly –
cofactors,
coenzymes,
prosthetic groups,
other modifications
Cofactor is a general term for functional non-amino acid component
Metal ions or organic molecules
Coenzyme is used to designate an organic cofactors
NAD+ in lactate dehydrogenase
Prosthetic groups are covalently attached cofactors
Heme in myoglobin

26. Polypeptide Size in Some Proteins

27. Classes of Conjugated Proteins

28. Peptides and Proteins- Burning Questions
Sequence and composition?
Three-dimensional structure?
Folding Mechanism?
Biochemical role?
Functional regulation?
Molecular interactions with small and macro-molecules?
Structural and sequence relatives?
Cellular and sub-cellular localization?
Physical and chemical properties?

29. Purification – Fractionation of Protein Mixtures
Separation relies on differences in physico-chemical properties
Solubility – Selective Precipitation (Centrifugation)
Thermal stability --
Charge --Electrophoresis, Isoelectric Focusing, IEC
Size – Dialysis, Sedimentation (Centrifugation), GFC
Affinity for a ligand – “Pull down” assays (Centrifugation), AC
Hydrophobicity (HIC)
Chromatography is commonly used for preparative separation

30. Protein Fractionation
FIGURE 3-16 Column chromatography. The standard elements of a chromatographic column include a solid, porous material (matrix) supported inside a column, generally made of plastic or glass. A solution, the mobile phase, flows through the matrix, the stationary phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is layered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separation improves (i.e., resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions.

31. Native gel electrophoresis Iso-electric Focusing
Separation by Charge
Ion Exchange Chromatography
Anion exchange
Matrix positive
Proteins negative
Displaced by anions
Cation exchange – Opposite
pH determines net charge on Proteins
Salt concentration gradient
Native gel electrophoresis
Iso-electric Focusing

32. Separation by Size Size exclusion (Gel Filtration) Chromatography
Loading vol. <5% of column volume
Samples diluted
Dialysis or Centrifugal concentrators

33. Separation by Affinity
Affinity Chromatography
Free Ligand-Beads -- centrifugation
Ligand-Magnetic-Beads
Immuno-assays on solid supports

34. Electrophoresis for Protein Analysis
Separation in analytical scale is commonly done by electrophoresis
Electric field pulls proteins according to their charge
Gel matrix hinders mobility of proteins according to their size and shape

35. SDS PAGE: Molecular Weight
SDS – sodium dodecyl sulfate – a detergent
SDS micelles binds to, and unfold all the proteins
SDS gives all proteins an uniformly negative charge
The native shape of proteins does not matter
Rate of movement will only depend on size: small proteins will move faster
In this case the effect of charge is eliminated by binding a negatively charged detergent, SDS, to all the proteins which are denatured.
The SDS binds uniformly per unit length of protein and therefore the force on the molecules from the field will be a uniform amount per unit length and the only affect on the speed of travel will be the retarding force due to their size.
This is therefore a method to separate molecules based on their molecular weights. Clearly not useful for oligomers since these will be forced apart by the SDS.

36. Protein Sequencing
Pehr Edman and Frederick Sanger

37. Spectroscopic Detection of Aromatic Amino Acids
The aromatic amino acids absorb light in the UV region
Proteins typically have UV absorbance maxima around nm
Tryptophan and tyrosine are the strongest chromophores
Concentration can be determined by UV-visible spectrophotometry using Beers law: A = ·c·l

38. Chapter 3: Summary In this chapter, we learned about:
The many biological functions of peptides and proteins
The structures and names of amino acids found in proteins
The ionization properties of amino acids and peptides
The methods for separation and analysis of proteins

39. Nonpolar, Aliphatic R Groups

40. Aromatic R Groups Also Hydrophobic
These amino acid side chains absorb UV light at nm

41. Polar, Uncharged R Groups
These amino acids side chains can form hydrogen bonding
Cysteine can form disulfide bonds

42. Basic R Groups

43. Acidic R Groups