Protein Structure

Henderson-Hasselbalch equation

2
Buffers Stabilize Against pH
Changes
Absence of buffer, pH 7.4=10-7.4 = 5 x 10-8 M
pH 7.3=10-7.3 = 4 x10-8 M
Difference of 1 x 10-8 M

With a 0.1 mM phosphate buffer 5600 times more

acid is consumed going from pH 7.4 to 7.3.

3
 What about [PO43-] and
[H3PO4]?
[PO43-] / [HPO42-] = 107.4-12.4 = 10-5.0

[H2PO4-] / [H3PO4] = 107.4-2.2 = 105.2

Neither form of phosphate is present in significant

quantities at pH 7.4.
Buffers are only effective within +/- 0.5 pH of their pKa.

4
Primary Structure, Summary
Amino acids are linked by peptide bonds.
Properties of peptide bonds:
Planar.

The order of amino acids determines structure.

Torsion angles limited by steric hindrance.
Visualized by Ramachandran plots.

5
The Peptide Bond is Planar

6
Peptide Bonds can be cis or trans

trans predominates

7
Proline is Almost Equally
Cis/Trans

8
The Caa Bonds Have Free
Rotation

9
Dihedral or torsion angles define the degrees of
rotation

: the angle betw. : the angle betw.

C-N-C-C N-C-C-N

10
Only Some Conformations are Allowed

11
The Amino Acid Sequence
Determines 2o and 3o Structures

PDB: 2A3G 12
Protein Secondary Structure
The local folds created from adjacent amino
acids in a polypeptide chain.

13
2 is Formed from Backbone H-Bonds

H-Bond Donor H-Bond Acceptor

14
The -helix
By far the most common 2 in proteins.
Same name as DNA helix, different structure.
Backbone inside/ side chain out portion
Left hand less stable more sterical hindrance

15
Backbone H-bonds in an -helix
Between every 4th amino acid.
=O - - - H-N.

16
 Backbone H-bonds Set the -helix Dimensions
There are 3.6 amino acids per turn.
Each residue is 1.5 farther up the helix,
and 100 from its nearest neighbour.

Each turn is thus 1.5 x 3.6 = 5.4

long.
This is known as the pitch.
5.4

17
 The Amino Acid R Groups Stick Out
A repeating pattern.
Useful for protein-protein interactions.

Jain and Tu. Int. J. Mol. Sci. 12, 1431-50. 2011. 18

-Helices can be Left- or Right-handed
Right-handed helices are far more
stable/common.

19
Some AA Destabilize Helices
Valine, threonine, and isoleucine are too bulky
to fit easily into helices.

Proline is a helix breaker.

The amide nitrogen cant H-bond.
The R-group cant adopt a suitable torsion angle.
20
Some AA Destabilize Helices,
Pt.2
Serine, aspartate, and asparagine can H-Bond
with the main chain.

This competes with the backbone interactions.

21
Some Proteins are Mostly -
helical
Ex. Ferritin, which forms a 4-helix bundle.

22
The b-Sheet
Less restricted in
conformation than
the -helix.

23
Each b-Strand is a Long Chain

24
-strands H-Bond with Each
Other
C-c-n-c-c
N-c-c-n-c

Anti-parallel b
sheet 25
-strands H-Bond with Each
Other

Parallel b
sheet 26
Mixed b-sheet Also Occur

27
b-sheets can be Flat or Twisted

28
A Circular b-sheet is a b-Barrel
Very common in
membrane pores.

Usually antiparallel.

29
Comparing -sheets and -helices
-sheet
Hydrogen bonds between strands, amino acids far
apart in primary sequence

-helix
Hydrogen bonds between amino acids close in
sequence

Both
Hydrogen bonds between NH and C=O in peptide
backbone

30
The b-turn is Small 2o Structure
Also called the hairpin turn or reverse turn.
Surface of the polypeptide= interact with the
polar solvent/molecules

31
Other Types of Loops: Long and Disordered
Usually on the protein
surface.
No common structure
or length.
Often involved in
protein binding.

Antibody Fragment

32
33
 Tertiary Structures
The combination of 2 structural elements into a
larger structure. In aqueous
Single protein.
Hydrophobic effect is the most important role in
helping polypeptide.
Vander vals, ionic int, hydrogen bond also help

Fig 18.8. Introduction to Chemistry, 1.0. 2011. 34

Complex 3 Strs. are Built From Structural
Motifs
Aka supersecondary structures.
Some are just common folding patterns, others serve
specific structural roles.

The Greek Key

35
 Many Motifs Contain Binding
Pockets

Zinc Finger Rossmann Fold

Binds zinc for DNA recognition Binds Nucleotides
36
Myoglobin: The First Known Protein Structure
A globular protein. Supplies oxygen to muscles.

45 35 25
37
Heme Uses Iron to Bind Oxygen
Built from just glycine and
acetate.

Can bind only 1 O2 each

Hemoglobin have 4 heme
so 4 o2
Myoglobin bring o2 inside
our muscle=tertiary

38
Distribution of AA in Myoglobin
Blue=Hydrophilic Yellow=Hydrophobic

Outside Inside
39
The Hydrophobic Effect

40
Gibbs free energy
G = H TS
If G < 0, reaction is favourable (spontaneous)

The hydrophobic effect maximizes H, with a minor

S penalty.
The result of H2O-H2O and H2O-protein hydrogen bonds.

41
Membranes Invert the Hydrophobic Effect
Polypeptide backbone in a hydrophobic
environment are stabilized by pairing all the NH
and CO groups in H- bonds
-helices or -sheets are favoured, because these
pair every backbone residue with a complementary
donor/acceptor.

-helices and -sheets can both form

hydrophobic and hydrophilic sides.

42
Membranes Invert the Hydrophobic Effect
Pt.2
Van der Waals interactions between adjacent
hydrocarbon side chains also contribute to
protein stability
With 20 amino acids of different sizes and
shapes the protein interior can be tightly
packed to maximize van der Waals interactions
Ionic interactions and H-bonds between side chains
are very strong in hydrophobic environments..

43
Distribution of AA in Membrane Proteins
The opposite of a
soluble protein.

44
Quaternary Structures
The combination of several polypeptide strands
into a larger structure.
4 subunits:

45
-Helical coiled-coil proteins
Long, strong fibrils.
Found in muscles (myosin) and hair (keratin).
1000 (100 nm, or 0.1 m) or longer

46
Coiled-coil Proteins Have a Characteristic heptad repeat

In a-keratin, every 7th residue is

leucine
Van der Waals interactions between
leucines stabilize the helix.
Covalent/non-covalent bond
Ionic
hydrogen

Artificial coiled-coil proteins can

be built from this pattern.

Arronsson et al. Sci. Rep. 5: 14063. 2015. 47

Keratin is Also Stabilized by Disulfide Bridges
A perm rearranges these crosslinks to create
kinks and bends in the strand.the more disulf
bond the stronger it is that moelcule

http://evoice.ewha.ac.kr/news/articleView.html?idxno=517
http://imgkid.com/loose-spiral-perms-before-and-after.shtml

48
Collagen Holds Human Cells Together
Main fibrous component of skin, bone, tendon,
cartilage, and teeth (extracellular).
3 helical polypeptide chains, each ~1000 residues long.

49
The Collagen Triple-Helix is
Unique
No H-bonds within strands!
Strands are stabilized by steric
repulsion between Pro and
Hydroxyproline (Hyp).

Glycine H-bonds hold the

strands together.
The smallest AA, it flexible enough
for a H-bond to form.

Hydroxyproline biosynthesis
requires vitamin C. 50
Glycine is Key to the Collagen Triple
Helix

Osteogenesis imperfecta (brittle

bone disease) occurs when any
other amino acids replace the
internal glycine residues. Improper
folding results, and defective
collagen accumulates.
51
What determines These 3D
Strs.?
The primary amino acid sequence.

52
Case Study: Bovine
ribonuclease
Degrades RNA
ase = degradative
enzyme.

4 disulfide bridges.

Very stable.

53
Two denaturants and a
reductant

Its oxydise and breaks down S-H bond

54
-Mercaptoethanol Reduces Disulfide
Bridges
A very mild oxidant.
This reversible process is driven by Le Chteliers
principle.
5 M protein, 10 mM -ME.
If removed but urine stays; doesnt go back to its initial form
but the disulfide bond get together bc its the 1er sequence
that give the structure and if its not sequence one by one by
order it fucks up everyhting

55
 Urea Disrupts H-Bonds

Enzymatically active Enzymatically inactive

56
Bovine Ribonuclease Can Spontaneously Refold

The sequence alone determines 3 structure.

Folding occurs on dilution in fresh buffer.

-
+O
2

This protein is structurally simple. Some more

complex proteins need protein chaperones to fold
correctly.
57
 Oxidation in Urea Scrambles the Disulfide Bonds
Enzymatically
With trace ME these misfolded inactive
proteins can then reform the
active structure.
The active structure has the lowest
G, making it the most
thermodynamically stable.
The 1er sequence structure of AA that dictate the proper
formation of the 3rd structure of that enzyme
If we keep the scrambled ribonuclease and add a trace of BME=
reform to the stable structure

Enzymatically
active 58
For the tutorial quiz week 1:
-be able to do pH problems (simple ones)
-ionization of groups on amino acids
-amino acids-be able to a) recognize structures; b) know
names, 1 letter code and 3 letter code; c) know properties
of R groups

For the laboratory week 1:

-read the intro to the lab manual
-come with required supplies and clothing
-prepare a pre-lab (see page 5 of the manual)