1. Lecture 13
February 16, 2016
Biotech 3

2. Sickle Cell Anemia Gln  Val
Normally a benign mutation.
No effects on the 2°, 3°, or 4° structures  of hemoglobin under normal oxygen concentration.
Under low oxygen concentration, hemoglobin exposes a hydrophobic patch.
The hydrophobic side chain of valine associates with the hydrophobic patch, causing hemoglobin to aggregate and form fibrous precipitates.

3. Amino Acid Residues Aliphatic Residues: G, A, V, L, I, P
amino acids with a open chain hydrocarbon side groups, not aromatic rings.
No reactive groups on side chain.
Do not interact favorably with water.
Interact favorably with each other (the “bricks” of a functional protein molecule)
Imposes rigidity constraints
Have effect on conformation of protein “backbone”
Promotes turns
Least hydrophobic
Most hydrophobic
Hydrophobicity

4. Amino Acid Residues Cont.
Charged Residues: D, E, H, K, R
(sometimes charged)
Negative Charge
Chain length confers differences in interactions
Interact with amines
Positive Charge

5. Amino Acid Residues Cont.2
Aromatic Residues: F, T, W

6. Aromatic Amino Acid Residue Absorption Spectrum
Aromatic Residues: F, T, W
Aromatic side chains are responsible for most of the UV absorbance and fluorescence.
Spectral properties are sensitive to immediate environment of side chain.
Useful to use as probes of protein structure.
Phe least reactive.
Hydroxyl group of Tyr is reactive.
Trp is less frequent in proteins.
Absorbance
Fluorescence
λmax
λmax
Phenylalanine
Tyrosine
Tryptophan
257.4
282
274.6
303
279.8
348

7. Amino Acid Residues - Histidine
Nitrogen with the hydrogen is an electrophile and donor for hydrogen bonding
Nitrogen without a hydrogen is a nucleophile and acceptor for hydrogen bonding
Very versatile
Can undergo numerous reactions
Ex) Phosphorylation

8. Amino Acid Residues – Sulfur
Sulfur Containing Residues: M and C
Most reactive of any amino acid side chain
Thiol group can complex with copper, iron, zinc, cobalt, molybdenum, manganese, cadmium, mercury, and silver.
Form disulfide bonds!
Codeed by the start codon; ATG
Relatively unreactive
Can be labeled
ex) methyl iodide

9. -S – CH2 – -S – CH2 – Oxidation of Cysteine – CH2 – S – S – CH2 –
Cysteine disulfide bond
Reduced
-S – CH2 –
Oxidized
“Cystine” residue
– CH2 – S – S – CH2 –
-S – CH2 –

10. -S – CH2 – -S – CH2 – Reduction of Cystine – CH2 – S – S – CH2 –
Cysteine disulfide bond
Reduced
-S – CH2 –
Oxidized
“Cystine” residue
– CH2 – S – S – CH2 –
Can be effectively reduced by thiol-disulfide exchange with thiol reagents
β-mercaptoethanol (BME)
Dithiothreitol (DTT)
Tris(2-carboxymethyl)phosphine (TCEP)
-S – CH2 –

11. Protein Electrophoresis – Reducing agent
a. Reducing agent
Reduces disulfide bonds
Separates multi-subunit proteins linked by disulfide bridges
β-mercaptoethanol (BME), dithiothreitol (DTT), TCEP

12. Protein Structure Proteins perform many functions
Structural support – cellular cytoskeleton
Metabolism – enzymes and hormones
Protection – antibodies
Communication – signal transduction
Regulation – transcription factors
Two main groups:
Fibrous – form cellular structures: keratin, myosin, collagen, etc
Globular – signaling and chemical changes within cell: enzymes

13. Protein Structure Cont.
The main driving force for folding water-soluble globular proteins is to pack hydrophobic side chains into interior of the molecule; form a hydrophobic core.
Problem: the main chain (aka backbone) is highly polar and therefore hydrophilic!
Solution: formation of secondary structures within the interior of the protein molecule.
Two types of secondary structures: alpha helices and beta sheets
Secondary structures are characterize by the hydrogen bonds between the main chain NH and C=O groups.
Hydrogen bond acceptor
Hydrogen bond donor

14. Alpha (α ) Helix in Protein Structures
Amino terminus
Spiral structure
May be more loosely or tightly coiled
3.6 residues per turn
May vary considerably in length
All hydrogen bonds in an α helix point in the same direction
Significant net dipole moment in α helix:
partial positive charge at amino end
partial negative charge at carboxy end
Preferred amino acids in α helix
Proline fits very well into the first turn of an α helix (may also form a bend)
Ala (A), Glu (E), Leu (L), Met (M)  Good
Pro (P), Gly (G), Tyr (Y), and Ser (S)  Poor
Typically on the outside of protein
May cross membranes (α helix will have more hydrophobic amino acids)
3.6 residues/turn
Dipole moment
Carboxyl terminus

15. Pi (π) Helix in Protein Structures
Spiral structure
May be more loosely or tightly coiled
3.6 residues per turn
May vary considerably in length
All hydrogen bonds in an α helix point in the same direction
Significant net dipole moment in α helix:
partial positive charge at amino end
partial negative charge at carboxy end
Preferred amino acids in α helix
Proline fits very well into the first turn of an α helix (may also form a bend)
Ala (A), Glu (E), Leu (L), Met (M)  Good
Pro (P), Gly (G), Tyr (Y), and Ser (S)  Poor
Typically on the outside of protein
May cross membranes (α helix will have more hydrophobic amino acids)

16. Helical Wheel Representation of Alpha Helices1.
Leu
Ser
Phe
Ala
Ala
Ala
Met
Asn
Gly
Leu
Ala 2.
Ile
Asn
Glu
Gly
Phe
Asp
Leu
Leu
Arg
Ser
Gly
hydrophobic
Charged
Polar 3.
Lys
Glu
Asp
Ala
Lys
Gly
Lys
Ser
Glu
Glu
Glu
Helical wheel representation of α helices:

17. Beta (β ) in Protein Structures
Built from several regions of the polypeptide chain into β-strands
5 – 10 residues per strand
Strands are aligned adjacent to each other into β-sheets:
Parallel: amino to carboxy terminal run in the same direction
Antiparallel: amino to carboxy terminal alternate direction
Mixed: combination of parallel and antiparallel (bias against mixed β-sheets)

18. Beta (β ) in Protein Structures - Parallel
Hydrogen bonds: main chain NH (amide from peptide bond) and O atoms within a β sheet

19. Beta (β ) in Protein Structures – Anti parallel
Hydrogen bonds: main chain NH (amide from peptide bond) and O atoms within a β sheet

20. Beta (β ) in Protein Structures – Hairpin Loop

21. β Sheet Topology
Topology diagrams representation of β sheets:

22. Protein Structure Motifs
Motif: simple combination of a few secondary structure elements with a specific geometric arrangement that are frequently found to occur in protein structures.
(tertiary structures)
Examples:
Helix-turn-helix
Hairpin Beta
Greek Key
Which amino acid would you expect to find in the ‘turn”

23. Protein Structure Continued
Structural hierarchy; primary, secondary, tertiary, quaternary.
Simple motifs combine to form complex motifs.
Large polypeptide chains fold into several domains (domains typically are also units of function).
Single domain or multiple domains.
Domains are built from structural motifs.
α domains
Β domains
α/β domains