1. Amino acids, Peptides, Proteins
Andy Howard
Introductory Biochemistry Fall 2013, IIT

2. Proteins are worth studying
We’ll finish our explanation of amino acid properties, and then move on to peptide and protein structures
09/03/2013
Amino Acids, Peptides, Proteins

3. Amino Acids, Peptides, Proteins
Plans
Amino acids, concluded
Peptides, proteins
The notion of protein structure
Levels of protein structure
Domains
TIM barrels
Generalizations about structure
09/03/2013
Amino Acids, Peptides, Proteins

4. Side-chain reactivity
Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups
Side chains can participate in reactions:
Acid-base reactions
Other reactions
In proteins and peptides, the side-chain reactivity is more important because the main chain is locked up!
09/03/2013
Amino Acids, Peptides, Proteins

5. Acid-base reactivity on side chains
Asp, glu: side-chain COO-:
Asp sidechain pKa = 3.9
Glu sidechain pKa = 4.1
That means that at pH = 5.1, a glutamate will be ~90.9% charged
Lys, arg: side-chain N:
Lys sidechain –NH3+ pKa = 10.5
Arg sidechain =NH2+ pKa = 12.5
09/03/2013
Amino Acids, Peptides, Proteins

6. Acid-base reactivity in histidine
It’s easy to protonate and deprotonate the imidazole group
09/03/2013
Amino Acids, Peptides, Proteins

7. Cysteine: a special case
The sulfur is surprisingly ionizable
Within proteins it often remains unionized even at higher pH
09/03/2013
Amino Acids, Peptides, Proteins

8. Amino Acids, Peptides, Proteins
Ionizing hydroxyls
X–O–H  X–O- + H+
Tyrosine is easy, ser and thr hard:
Tyr pKa = 10.5
Ser, Thr pKa = ~13
Difference due to resonance stabilization of phenolate ion:
09/03/2013
Amino Acids, Peptides, Proteins

9. Resonance-stabilized ion
09/03/2013
Amino Acids, Peptides, Proteins

10. Other side-chain reactions
Little activity in hydrophobic amino acids other than van der Waals
Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, …
Nitrogens in his can covalently bond to various ligands
Hydroxyls can form ethers, esters
Salt bridges (e.g. lys - asp)
09/03/2013
Amino Acids, Peptides, Proteins

11. Amino Acids, Peptides, Proteins
Phosphorylation
ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr
ATP + Ser-OH  ADP + Ser-O-(P)
Often involved in activating or inactivating enzymes
Under careful control of enzymes called kinases and phosphatases
This is an instance of post-translational modification
09/03/2013
Amino Acids, Peptides, Proteins

12. Amino acid frequencies and importance in active sites
Polar amino acids, particularly S, H, D, E, K, are at the heart of most active sites of enzymes and other globular proteins
Yet they’re relatively uncommon overall in proteins
Nonpolar amino acids (V, L, I, A) occur with higher frequencies overall
09/03/2013
Amino Acids, Peptides, Proteins

13. Amino Acids, Peptides, Proteins
Peptides and proteins
Peptides are oligomers of amino acids
Proteins are polymers
Dividing line is a little vague: ~ aa.
All are created, both formally and in practice, by stepwise polymerization
Water eliminated at each step
09/03/2013
Amino Acids, Peptides, Proteins

14. Growth of oligo- or polypeptide
09/03/2013
Amino Acids, Peptides, Proteins

15. Amino Acids, Peptides, Proteins
The peptide bond
The amide bond between two successive amino acids is known as a peptide bond
The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character
09/03/2013
Amino Acids, Peptides, Proteins

16. Double-bond character of peptide
09/03/2013
Amino Acids, Peptides, Proteins

17. Amino Acids, Peptides, Proteins
The result: planarity!
This partial double bond character means the nitrogen is sp2 hybridized
Six atoms must lie in a single plane:
First amino acid’s alpha carbon
Carbonyl carbon
Carbonyl oxygen
Second amino acid’s amide nitrogen
Amide hydrogen
Second amino acid’s alpha carbon
09/03/2013
Amino Acids, Peptides, Proteins

18. Rotations and flexibility
Planarity implies  = 180º, where  is the torsion angle about N-C bond
Free rotations are possible about N-C and C-C bonds
Define  = torsional rotation about N-C
Define  = torsional rotation about C-C
We can characterize main-chain conformations according to , 
09/03/2013
Amino Acids, Peptides, Proteins

19. Amino Acids, Peptides, Proteins
Ramachandran angles
G.N. Ramachandran
09/03/2013
Amino Acids, Peptides, Proteins

20. Preferred Values of  and 
Steric hindrance makes some values unlikely
Specific values are characteristic of particular types of secondary structure
Most structures with forbidden values of  and  turn out to be errors
09/03/2013
Amino Acids, Peptides, Proteins

21. Amino Acids, Peptides, Proteins
How far from 180º can w vary?
Remember what we said about the partial double bond character of the C-N main-chain bond
That imposes planarity
In practice it rarely varies by more than a few degrees (< 5º) from 180º.
Aromatic amino acids (F, Y, W) are the most likely to depart from planarity
09/03/2013
Amino Acids, Peptides, Proteins

22. Amino Acids, Peptides, Proteins
Ramachandran plot
Cf. figures in text
If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them!
09/03/2013
Amino Acids, Peptides, Proteins

23. How are oligo- and polypeptides synthesized?
Formation of the peptide linkages occurs in the ribosome under careful enzymatic control
Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine):
GTP + n-length-peptide + amino acid  GDP + Pi + (n+1)-length peptide
09/03/2013
Amino Acids, Peptides, Proteins

24. What happens at the ends?
Usually there’s a free amino end and a free carboxyl end:
H3N+-CHR-CO-(peptide)n-NH-CHR-COO-
Cyclic peptides do occur
Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step.
09/03/2013
Amino Acids, Peptides, Proteins

25. Reactivity in peptides & proteins
Main-chain acid-base reactivity unavailable except on the ends
Side-chain reactivity available but with slightly modified pKa values.
Terminal main-chain pKavalues modified too
Environment of protein side chain is often hydrophobic, unlike free amino acid side chain
09/03/2013
Amino Acids, Peptides, Proteins

26. Amino Acids, Peptides, Proteins
iClicker question
1. What’s the net charge on ELVIS at pH 7?
(a) 0
(b) +1
(c) -1
(d) +2
(e) -2
09/03/2013
Amino Acids, Peptides, Proteins

27. Amino Acids, Peptides, Proteins
… and one more:
2. An amino acid within a protein has main-chain torsion angles =-90, =-60. This amino acid is probably
(a) part of a right-handed -helix
(b) part of a -sheet
(c) part of a left-handed -helix
(d) outside of any secondary structure
(e) none of the above
09/03/2013
Amino Acids, Peptides, Proteins

28. Amino Acids, Peptides, Proteins
Disulfides
In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains
09/03/2013
Amino Acids, Peptides, Proteins

29. Amino Acids, Peptides, Proteins
What could this do?
Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another
This can influence protein stability
09/03/2013
Amino Acids, Peptides, Proteins

30. What does it mean to characterize a molecule’s structure?
In general, covalent bonds are of constant length and produce constant bond angles
Single covalent bonds, though, allow for rotation about the bond
Therefore the 3-D structure can either be floppy or well-defined
Peptides and noncyclic sugars flop (mostly)
Proteins: mostly well-defined; a little flexibility on the surface
09/03/2013
Amino Acids, Peptides, Proteins

31. Proteins have well-defined structures
This is not necessarily obvious!
Many biopolymers, notably polysaccharides and to some degree polynucleotides, are floppier
Proteins do have some flexibility, particularly near their surfaces, but they behave approximately like rigid bodies under many circumstances.
1E7H
09/03/2013
Amino Acids, Peptides, Proteins

32. Protein Structure Helps us Understand Protein Function
If we do know what a protein does, its structure will tell us how it does it.
If we don’t know what a protein does, its structure might give us what we need to know to figure out its function.
09/03/2013
Amino Acids, Peptides, Proteins

33. Levels of Protein Structure: G&G §5.1
We conventionally describe proteins at four levels of structure, from most local to most global:
Primary: linear sequence of peptide units and covalent disulfide bonds
Secondary: main-chain H-bonds that define short-range order in structure
Tertiary: three-dimensional fold of a polypeptide
Quaternary: Folds of multiple polypeptide chains to form a complete oligomeric unit
09/03/2013
Amino Acids, Peptides, Proteins

34. What does the primary structure look like? (G&G §5.3)
-ala-glu-val-thr-asp-pro-gly- …
Can be determined by amino acid sequencing of the protein
Can also be determined by sequencing the gene and then using the codon information to define the protein sequence
Amino acid analysis means percentages; that’s less informative than the sequence
09/03/2013
Amino Acids, Peptides, Proteins

35. Components of secondary structure (G&G §6.3)
, 310,  helices
pleated sheets and the strands that comprise them
Beta turns
More specialized structures like collagen helices
09/03/2013
Amino Acids, Peptides, Proteins

36. An accounting for secondary structure: phospholipase A2
09/03/2013
Amino Acids, Peptides, Proteins

37. Amino Acids, Peptides, Proteins
Alpha helix (G&G fig. 6.6)
09/03/2013
Amino Acids, Peptides, Proteins

38. Characteristics of  helices (G&G Fig. 6.9)
Hydrogen bonding from amino nitrogen to carbonyl oxygen in the residue 4 earlier in the chain
3.6 residues per turn
Amino acid side chains face outward, for the most part
~ 10 residues long in globular proteins
09/03/2013
Amino Acids, Peptides, Proteins