Module 2: Protein structure
Learning Outcomes
•
Associate Professor Terry Mulhern
1
Learning Outcomes
1.
2.
3.
4.
5.
6.
7.
8.
9.
11.
12.
13.
Describe where you would expect to find polar and nonpolar amino
acids in a folded globular protein.
Describe were you would expect to find Gly and Pro in a folded protein.
List the overall features of folded proteins.
Explain why protein folding is said to be cooperative.
Explain how Christian Anfinsen’s experiments showed that under
appropriate conditions protein folding is reversible.
Describe the role of disulfide bonds in protein folding.
Describe how cellular conditions are not ‘ideal’ for protein folding.
Explain the role of protein folding chaperones in ‘protecting’ unfolded
proteins from ‘misfolding’.
List the forces drive protein folding and which chemical groups and
amino acid type are involved in each interaction.
Explain the thermodynamic basis of the hydrophobic interaction. List the
different regions of a Ramachandran plot.
Draw a labelled Ramachandran plot.
Interpret structural information from a Ramachandran plot.
Learning Outcomes
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Describe how hydrogen bonding helps make proteins compact.
Identify different hydrogen bonding interactions in a protein
List the structural properties of alpha-helices.
Explain why alpha-helices are often ‘amphipathic’.
Draw a simplified helical wheel diagram.
Read amino acid sequences to identify heptad repeat patterns.
Explain the difference between a beta-strand and a beta-sheet.
List the structural properties of beta-sheets.
Explain how a beta-sheet can have hydrophilic and hydrophobic face.
Read amino acid sequences to identify alternating sequence patterns.
Draw a diagrams illustrating hydrogen bonding in antiparallel and parallel betasheets.
26. List the structural properties of reverse turns.
27. Explain the difference between type-I and type-II turns.
28. Read amino acid sequences to identify where turns are likely.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Learning Outcomes
Define regular and irregular structures.
Identify regular and irregular structures from structural information.
List the structural properties of proteins.
Describe the three common supersecondary structures.
Identify supersecondary structure in images of proteins.
Explain the difference between primary, secondary, tertiary and quaternary
protein structure.
From images of proteins, identify secondary, tertiary and quaternary protein
structures.
Explain the difference between the terms domain fold and module.
Define domain in terms of structure and evolution.
Describe the genetic processes that create proteins with different functional
properties using existing domain structures.
Explain what is meant by the statement that protein sequences can be
optimally aligned.
Explain the link between sequence identity, ancestry, and structural similarity.
Define homologue, orthologue and paralogue.
Module 2: Protein structure
Video 1: Locations of
amino acids in
proteins
•
Associate Professor Terry Mulhern
5
What is Expected of You?
Learning Outcomes:(1)
(2)
Describe where you would expect to find polar and nonpolar amino
acids in a folded globular protein.
Describe were you would expect to find Gly and Pro in a folded
protein.
Location of residue types
Where are different residues found?
Example: Lysozyme
All heavy
atoms
• There is no empty space inside a
protein (proteins are compact)
• Water is excluded from inside
proteins.
ribbon
heavy
side chain
atoms
Heavy atoms: C,O,N,S
Carbon: Black
Oxygen: Red
Nitrogen: Blue
Sulfur: Yellow
Location of residue types
Where are different residues found?
Example: Lysozyme
Polar side chains
Exposed on the surface
Nonpolar side chains
Buried in the core
Location of residue types
Where are different residues found?
Example: Lysozyme
Gly
Backbone shown
(no side chain)
In turns
Pro
In turns
Module 2: Protein structure
Video 2: Protein folding,
unfolding and re-folding
•
Associate Professor Terry Mulhern
10
What is Expected of You?
Learning Outcomes:-
(1) List the overall features of folded proteins.
(2) Understand why protein folding is said to be
cooperative.
(3) Explain how Christian Anfinsen’s experiments showed
that under appropriate conditions protein folding is
reversible.
(4) Describe the role of disulfide bonds in protein folding
What forces drive protein folding?
General features of folded protein structures
•Proteins are compact: there tends to be no empty space inside
proteins because of close packing of backbone and side chain atoms
•Water is generally excluded from the interior of proteins
•Nonpolar (hydrophobic) side chains are usually located inside the
protein
•Polar (hydrophilic) side chains are usually located on the outside of
the protein
•How does a regular structure like the backbone pack closely?
•How do irregular structures like side chains packing closely?
•Why do proteins fold?
i.e Why is the folded state more stable than unfolded states?
What forces drive protein folding?
•electrostatic forces
•van der Waals interactions
•hydrogen bonds
•hydrophobic interactions
These interactions combine to stabilise the folded state making it favoured
over the unfolded state
Folded state is called the ‘native’ state
Unfolded state is said to be ‘denatured’
In solution, an unfolded polypeptide with spontaneously fold up.
e.g. rapidly dilute a protein from denaturant (e.g. concentrated urea) to buffer.
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
Protein unfolding
Urea
To unfold a protein: ‘denature’ the native state
To refold a protein: ‘renature’
Figure 3-6a Molecular Biology of the Cell (© Garland Science 2008)
Protein folding/unfolding
pH, Temperature, Denaturants
Protein folding is co-operative (all or none)
Transition between two states: folded and unfolded
Folded and unfolded states are in equilibrium
Protein folding is reversible
% Unfolded
100%
Unfolded
50%
Folded
0%
6
8
10
pH
12
35
45
55
ºC
65
0
4
6
[GdnHCl]
8
Protein folding is cooperative
Generally, if any part of a protein fold is disrupted interactions with the
rest of the protein structure are disrupted and the remainder of the
structure will be lost. Conditions that disrupt any part of the structure will
lead to the whole protein unraveling.
Folded state is only marginally more stable than the unfolded state
Protein
folding/unfolding
is reversible
(Under the
appropriate
conditions)
https://en.wikipedia.org/wiki/Christian_B._Anfinsen
Protein unfolding
Protein re-folding
SH
HS
SH
HS
SH
HS
Remove
β-mercaptoethanol
SH
SH
Remove
8 M urea
Protein re-folding
Remove
8 M urea
Remove
β-mercaptoethanol
Protein re-folding
Oxidation in the presence of urea gave
‘mixed’ disulfides (scrambled). Only a
small fraction would have the pairings
correct (105 different ways to arrange 8
cysteines). Only the correct pairing can
stabilize the native structure.
Adding a trace of βME reduces
scrambled disulfides and then the
protein can refold correctly.
Under appropriate conditions
Protein folding/unfolding
is reversible
Protein re-folding
remove
β-mercaptoethanol
HS
Air
Oxidation
SH
HS
SH
HS
SH
HS
SH
SH
Remove
8 M urea
SH
HS
SH
HS
SH
HS
SH
Add a little
β-mercaptoethanol
Protein folding is reversible
(all the ‘information’ is in the sequence)
Disulfide bonds don’t ‘direct’ folding
Folding directs disulphide bond formation
Disulfide bonds increase the relative
stability of the folded state over the
unfolded state(s)
(lock on the correct folded state)
Module 2: Protein structure
Video 3: Chaperones
•
Associate Professor Terry Mulhern
24
What is Expected of You?
Learning Outcomes:(1)
(2)
Describe how cellular conditions are not ‘ideal’ for protein folding
Explain the role of protein folding chaperones in ‘protecting’ unfolded
proteins from ‘misfolding’
Protein folding in vivo: Chaperones
Although a polypeptide ‘should’ be able to fold unassisted
under ‘ideal’ solution conditions.
Conditions in the cell can make folding slow or impossible.
Molecular crowding: cells are highly concentrated solutions of
proteins/nucleic acids/sugars/lipids etc. ‘Inappropriate’
interactions may occur with other molecules before the
protein can fold.
Protein folding in vivo: Chaperones
https://mgl.scripps.edu/people/goodsell/illustration/public/
Protein folding in vivo: Chaperones
Although a polypeptide ‘should’ be able to fold unassisted, conditions in the cell can make folding slow
or impossible
Molecular crowding: cells are highly concentrated solutions
‘Inappropriate’ interactions may occur with other proteins/sugars/lipids etc. before the protein can fold
Nascent polypeptide may misfold as they comes off the ribosome. The
polypeptide chain grows by sequential addition of amino acid residues to the
C-terminal end of the chain. Misfolding may occur because the sequence is
not complete.
Chaperones don’t fold the protein –they help avoid misfolding. Chaperones
assist folding by binding to unfolded/partially folded polypeptides and
protecting them from misfolding. Chaperones bind to temporarily exposed
hydrophobic regions preventing them from interacting with the wrong partners:
‘inappropriate’ interactions.
Protein folding in vivo: Chaperones
https://mgl.scripps.edu/people/goodsell/illustration/public/
Module 2: Protein structure
Video 4: Forces that
drive protein folding
•
Associate Professor Terry Mulhern
31
What is Expected of You?
Learning Outcomes:(1)
(2)
List the forces drive protein folding and which chemical groups and
amino acid type are involved in each interaction.
Be able to explain the thermodynamic basis of the hydrophobic
interaction.
Favorable Interactions in Proteins
• Electrostatic interactions
– long-range strong interactions between permanently charged groups
– Salt bridges, especially those buried in the hydrophobic environment,
strongly stabilize the protein.
• London dispersion (van der Waals interactions)
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein.
• Hydrogen bonds
– Interaction of N−H and C=O of the peptide bond leads to local regular
structures such as α helices and β sheets.
• Hydrophobic effect
– The release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy.
Electrostatic forces: point charges
Ionic interactions between oppositely charged groups in proteins are called
“Salt bridges”
Charges tend to be distributed over several atoms
Component due to electrostatic interaction
Component due to hydrogen bonding
H
N
H
C
N
CH2
CH2
H
-
+
N
O
H
C
CH2
O
H
CH2
Arg
Glu
CH2
Electrostatic forces: dipoles
Molecules do not need net charge to form electrostatic interactions
Electron density can be localised due to electronegativity
(electronegativity O>N>C>S>H)
O
O
C
Cα
:N
H
-
C
Cα
Cα
-0.42
O
Example of dipole: Peptide bond
The peptide bond has partial double-bond
character due to resonance
Cα
+0.42
N
H+
Cα
C
Cα
N
Double bonded species is populated 40%
H
Giving rise to partial charges on the atoms
Separation of charges gives a “dipole moment” (distance×charge)
Dipoles can interact with each other and point charges (see α-helix)
-0.20
+0.20
Van der Waals interaction
Lennard-Jones
C12 C 6
E
r
=
−
(
)
6,12 potential
r 12 r 6
C12, C6 constants
0.2
Interaction
Energy
(kcal/mol)
Zero point at combined VDW radii
(Same interaction energy
as when separate)
Repulsion
Steeper 1/r12
0
∆E: Energy
Difference -0.2
Optimal VDW
Er - E∞
interaction
0
2
Attraction
Shallower 1/r6
4
6
Distance r (Å)
8
+ve inter. energy
Less favourable than
When ∞ separated
-ve inter. energy
More favourable than
When ∞ separated
Most favourable
distance
-almost touching
Hydrogen bonds
A hydrogen bond occurs when two electronegative atoms compete for
The same hydrogen atom:
Hydrogen bond
Acceptor
D
H
A
Hydrogen bond
Donor
δ−
δ+
δ−
D
H
A
The main component of the hydrogen bond is electrostatic
Dipole of D-H interacting with the partial negative charge on A
In strong hydrogen bonds there is also a covalent component
based on transfer of electrons from A to H
Result: the most favourable (and common) geometry has D-H..A collinear
Hydrophobic interactions
The electrostatic, hydrogen bond, and van der Waals interactions between
two polar groups in aqueous environment not particularly favourable
-because comparable competing interactions are possible with water
Why then do proteins fold?
Proteins also contain nonpolar groups.
Water is a poor solvent for nonpolar groups compared with organic solvents
Nonpolar groups cannot form hydrogen bond networks
Nonpolar groups prefer to interact with other nonpolar groups
The process is driven by entropy
∆G = ∆H − T∆S
Free energy
Enthalpy
(heat)
Entropy
(disorder)
Favourable ∆G (negative) given by negative ∆H + positive ∆S
i.e. increase the disorder somehow (but how?)
BUT Polypeptide chain is more ordered when it folds??
Hydrophobic interactions
Removal of hydrophobic side chains from water. Releases
ordered water from hydrophobic side chains
(favorable entropy)
E.g. oil droplets, organic
solvents in water etc.
The hydrophobic effect
Hydrophobic interactions
Removal of hydrophobic side chains from water
releases ordered water from hydrophobic side chains
Folded protein: Burial of
nonpolar side chians, many
‘ordered’ water molecules
released -disordered
Lower entropy
Unfolded
Exposed nonpolar side chains
Many ‘ordered’ water molecules
Higher entropy
Module 2: Protein structure
Video 5:
Ramachandran plots
•
Associate Professor Terry Mulhern
41
What is Expected of You?
Learning Outcomes:-
(1) List the different regions of a Ramachandran plot.
(2) Draw a labelled Ramachandran plot.
(3) Interpret structural information from a
Ramachandran plot.
Dihedral/torsion angles: φ and ψ
Why are phi (φ) and psi (ψ) so important?
Because: assuming all peptide bonds are trans
All the conformational freedom in the backbone of a polypeptide
is due to these two rotations. Everything else in this diagram is fixed
H
R
H
O
φ
C
Cα
N
H
C
N
ψ
C
Cα
O
The backbone conformation of a protein can be completely described
In terms of a pair of angles (φ,ψ)
Ramachandran plot
Named after the
Indian Biophysicist
G.N. Ramachandran
1968
Figure 3-3b Molecular Biology of the Cell (© Garland Science 2008)
The Polypeptide Is Made Up of a Series
of Planes Linked at α Carbons
Distribution of φ and ψ Dihedral Angles
• Some φ and ψ combinations are very unfavorable because of
steric crowding of backbone atoms with other atoms in the
backbone or side chains.
• Some φ and ψ combinations are more favorable because of
chance to form favorable H-bonding interactions along the
backbone.
• A Ramachandran plot shows the distribution of φ and ψ
dihedral angles that are found in a protein:
• shows the common secondary structure elements
• reveals the presence unusual backbone structure
Ramachandran Plot
Ramachandran Plot
Ramachandran plot for the enzyme
pyruvate kinase (isolated from rabbit)
are overlaid on the plot of
theoretically allowed conformations
Ramachandran Plot
• Not all residues in the ‘β’
region are in beta-strands
Regions of
‘Ramachandran space’
α
β
L
D
β
L
alpha
beta
left-handed turn
disallowed
• Not all residues in the ‘α’
region are in beta-strands
• ‘Disallowed’ really means
unfavourable or uncommon,
but not impossible
α
D
(all the white)
• All residues are subject to
steric hindrance that favours
the α, β or L regions –even if
they are in irregular structures
like ‘random coil’
Secondary Structures
• Secondary structure refers to a local spatial arrangement of the
polypeptide backbone.
• Two regular arrangements are common:
• the α helix
– Multiple consecutive residues in α region
– stabilized by hydrogen bonds between residues nearby in the sequence
• the β sheet
– Multiple consecutive residues in β region
– stabilized by hydrogen bonds between adjacent segments that may not be
nearby in the sequence
• Irregular arrangement of the polypeptide chain is called the random coil.
Module 2: Protein structure
Video 6: Alpha-helices
•
Associate Professor Terry Mulhern
51
What is Expected of You?
Learning Outcomes:-
(1) Describe how hydrogen bonding helps make
proteins compact
(2) Identify different hydrogen bonding interactions in a
protein
(3) List the structural properties of alpha-helices
(4) Explain why alpha-helices are often ‘amphipathic’
(5) Draw a simplified helical wheel diagram
(6) Read amino acid sequences to identify heptad
repeat patterns
Hydrogen bonds in proteins
• Any polar group buried in
the protein must form a
hydrogen bond. This will
include charged groups, but
these are rarely buried.
• There is a +ve partial
charge on the donor (H)
and –ve partial charge
on the acceptor (O).
−δ
+δ
+δ
−δ
• The atoms of a hydrogen bond can approach much closer
than a VDW interaction (2.7 Å compared to 1.9 Å) due to
covalent character of the hydrogen bond
• This increases the compactness and stability of a protein.
Hydrogen bonds: backbone and side chain
Can be:
Backbone-backbone
Backbone-side chain
Side chain-side chain
Hydrogen bonds: secondary structure
Close packing of the polypeptide backbone
Is achieved by several hydrogen bonding patterns
Beta-sheet (NH residue i to C=O residue j)
Alpha-helix (NH residue i to C=O residue i-4)
Reverse turns (NH residue i+3 to C=O residue i) L/R-handed
β
L
α
Hydrogen bonds should
Always be described
Donor to acceptor
NH to C=O
Hydrogen bonds: alpha-helix
N
C
3.6 residues per turn (100 ° per res.)
5.4 Å per turn (1.5 Å axial rise per res)
Side chains project outwards from helix axis
Hydrogen bonds: alpha-helix
N
RH
i-4
i-3
• The helix is right-handed
• Abundant
i-2
• φ,ψ -57º, -47º
• NH (residue i) to C=O (residue i-4)
• All NH and C=O within the helix
(except four at each end) form
favourable internal hydrogen bonds.
i-1
i
C
• Peptide bond dipoles add together
giving a macrodipole
N
O
||
C
i-4
H
|
N
i-3
i-2
i-1
i
i+1
i+2
i+3
i+4
C
Secondary structure: alpha-helices
Ways of forming a hydrophobic ‘core’
All α Bundles
Mixed αβ
Coiled coils
The α Helix: Top View
• The inner diameter of the helix (no side chains) is
about 4–5 Å.
• too small for anything to fit “inside” (not even
water)
• The outer diameter of the helix (with side chains) is
10–12 Å.
• happens to fit well into the major groove of dsDNA
• Amino acids #1 and #8 align nicely on top of each
other.
• What kind of sequence gives an α helix with one
hydrophobic face?
Secondary structure: alpha-helices
8
4
1
a
d
e
7 g
5
b 2
c
3
f
6
Heptad repeat: abcdefg
One heptad (7 x 100°) ~ 2 x 360°
Two times around
abcdefgabcdefgabcdefgabcdefg
abcdefgabcdefgabcdefg
| |
| |
| |
HxxHxxxHxxHxxxHxxHxxx
Sequence Affects Helix Stability
• Not all polypeptide sequences adopt α-helical structures.
• Small hydrophobic residues such as Ala and Leu are strong
helix formers.
• Pro acts as a helix breaker because it lacks the NH
hydrogen bond donor
• Gly acts as a helix breaker because the tiny R group
doesn’t contribute to stability of helix
• Attractive or repulsive interactions between side chains 3
to 4 amino acids apart will affect formation (e.g. stabilized
by oppositely charged residues 3-4 away in sequence)
Module 2: Protein structure
Video 7: Beta-sheets
•
Associate Professor Terry Mulhern
63
What is Expected of You?
Learning Outcomes:-
(1) Explain the difference between a beta-strand and a
beta-sheet
(2) List the structural properties of beta-sheets
(3) Explain how a beta-sheet can have hydrophilic and
hydrophobic face
(4) Read amino acid sequences to identify alternating
sequence patterns
(5) Draw a diagrams illustrating hydrogen bonding in
antiparallel and parallel beta-sheets
Beta-Sheets
• The planarity of the peptide bond and tetrahedral
geometry of the α carbon create a pleated sheet-like
structure.
• Sheet-like arrangement of the backbone is held together
by hydrogen bonds between the backbone amides in
different strands.
• Side chains protrude from the sheet, alternating in an
up-and-down direction.
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Hydrogen bonds: beta-sheet
Hydrogen bonds: beta-sheet
• A β-sheet consists of two or
more β-strands. The strand is
the element.
• Strands are either parallel or
antiparallel to neighbouring
strands. Sheets can be pure
or mixed.
• φ,ψ -130º, +130º (approx.)
• Twisted sheets are abundant
Parallel: H-bond
connect each residue to
two different residues
on opposite strand
Antiparallel: H-bond
connect each residue to
a single residue on
opposite strand
• Hydrogen bonds between NH
and C=O of neighbouring
strands: antiparallel more
stable; outer NH and C=O of a
sheet are not hydrogen
bonded.
In parallel β sheets, the H-bonded strands run in the same direction.
•
Hydrogen bonds between strands are bent (weaker).
In antiparallel β sheets, the H-bonded strands run in opposite directions.
• Hydrogen bonds between strands are linear (stronger).
Secondary structure: beta-sheet
e.g. β-barrel
Hydrophobic side chains are
packed into the core of the
barrel
Hydrophilic side chains
project outwards into the
solvent
Alternate residues
hydrophobic/hydrophilic
Module 2: Protein structure
Video 8: Reverse turns
•
Associate Professor Terry Mulhern
72
What is Expected of You?
Learning Outcomes:-
(1) List the structural properties of reverse turns
(2) Explain the difference between type-I and type-II
turns
(3) Read amino acid sequences to identify where turns
are likely
(4) Define regular and irregular structures
(5) Identify regular and irregular structures from
structural information
Secondary structure: beta-sheet
e.g. β-barrel
Hydrophobic side chains are
packed into the core of the
barrel
Hydrophilic side chains
project outwards into the
solvent
Alternate residues
hydrophobic/hydrophilic
Reverse Turns (β Turns)
• Turns occur frequently whenever strands in β sheets
change the direction.
• The 180° turn is accomplished over four amino acids.
• The turn is stabilized by a hydrogen bond from a
carbonyl oxygen of position 1 to amide hydrogen of
position 4 in the turn (i and i+3).
• Proline in position 2 (i+1) or glycine in position 3 (i+2)
are common in β turns.
Hydrogen bonds: beta-turns
 Called β-turns or reverse turns
 β-turns reverse the direction of the main chain
 Abundant, mostly on surface, redirect backbone
(particularly for β-sheets).
 Consist of four residues
 Residues i+1 and i+2 have different φ,ψ angles
 A hydrogen bond between NH of the fourth (i+3)
the carbonyl of the first (i) and the stabilizes the
turn.
 Proline often in position i+1 (φ=-60 matches
requirement of most β-turns)
 Type II turns often have Gly in position i+2,
R
Gly fav. because lack of side chain lessens “steric
hindrance” [C=O clash with R group]. Positive φ at
position i+2
 Type I and II differ by the direction i+1 carbonyl
Regular (and irregular) elements of protein structure
• β-sheets and α-helices are abundant elements of regular secondary
structure.
• Regular structure is defined as residues that have repeating φ,ψ angles
(consecutive residues).
• Regular structures are stabilized by a repeating pattern of hydrogen bonds.
They present side chains in a predictable fashion and pack together in
limited ways.
• β-turns are a simple way of reversing the main chain. They are also
stabilized by a hydrogen bond. Each residue in the turn performs a different
structural role and has different φ,ψ angles (irregular)
• Irregular structure often links regular elements and is termed ‘loop’ or
‘random coil’ structure
Why are we alive?
Various interactions stabilise the structure of proteins
Electrostatics: salt bridges
dipole interactions
Van der Waals: close packing of atoms
Hydrogen bonds:
side chain to side chain
side chain to backbone
backbone to backbone
Several backbone-backbone hydrogen bonding patterns facilitate close
packing of the polypeptide
These intra-protein effects in the folded state are almost balanced by
protein-solvent effects in the unfolded state; i.e. these forces alone are
not sufficient to make folded proteins stable
BUT
The burial of nonpolar groups in the hydrophobic core of proteins can
only occur in the folded state
Burial is entropically favourable (release of ordered water) and drives
protein folding
Because we are wet
Module 2: Protein structure
Video 9:
Supersecondary
structure
•
Associate Professor Terry Mulhern
80
What is Expected of You?
Learning Outcomes:-
(1) List the structural properties of proteins
(2) Describe the three common supersecondary
structures
(3) Identify supersecondary structure in images of
proteins
Principles of Protein Structure
• The peptide bond is usually trans, planar and fairly rigid.
• There are limitations on the dihedral angles that the main chain of
the protein can adopt (Ramachandran Plot).
• Repeating preferred φ,ψ angles are characteristic of the
commonly observed α-helices and β-strands.
• All buried polar groups to form hydrogen bonds.
• Proteins are compact because of favourable VDW contacts and
hydrogen bonds.
• There are a small group of supersecondary structure elements
that pack together to make up folded proteins
• There is an entropic requirement to bury hydrophobic residues
Most folds consist of a high content of three small repeating
supersecondary structural elements.
αα-hairpin
ββ-hairpin
β-trefoil
Jelly Roll
Immunoglobulin-like
TIM barrel
Ferredoxin-like
4-helix Updown
OB fold
UB-roll
Globin-like
Doubly wound
All folds
βαβ
Supersecondary
Structure (%)
83
47
67
82
38
90
77
55
88
68
62
αα-hairpin
ββ-hairpin
βαβ
Module 2: Protein structure
Video 10: Hierarchy of
protein structure
•
Associate Professor Terry Mulhern
85
What is Expected of You?
Learning Outcomes:-
(1) Explain the difference between primary, secondary,
tertiary and quaternary protein structure.
(2) From images of proteins, identify secondary, tertiary
and quaternary protein structures.
Four Levels of Protein Structure
αα-hairpin
ββ-hairpin
βαβ
Hierarchy of Protein Structure
subunit = polypeptide chain
1. Primary structure: amino acid sequence
2. Secondary structure: elements, α-helix, β-sheet, turns
(simple motifs α−α, β−β, β−α−β –supersecondary structure)
3. Tertiary structure (domains, folds, modules)
Primary structure
Nter-EEWYFGKITRRESERLLLNAENPRGTFLVRES -Cter
Secondary structure
Supersecondary structure
β1
β2
α
β−α−β
β1
α
β2
SH2
Tertiary structure
Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a protein.
• Stabilized by numerous weak interactions between amino
acid side chains
− largely hydrophobic and polar interactions
− can be stabilized by disulfide bonds
• Interacting amino acids are not necessarily next to each
other in the primary sequence.
• Tertiary structure refers to domains/folds/modules and
arrangement of domains (in a single subunit)
Hierarchy of Protein Structure
1. Primary structure: amino acid sequence
2. Secondary structure: elements, α-helix, β-sheet, turns
(simple motifs α−α, β−β, β−α−β –supersecondary structure)
3. Tertiary structure: folds, modules, domains, arrangement.
Nter-EEWYFGKITRRESERLLLNAENPRGTFLVRES -Cter
3
β−α−β
β1
α
β2
2
SH2
3
Quaternary Structure
A quaternary structure is formed by the assembly of
individual polypeptides into a larger functional cluster.
Hierarchy of protein structure
• Special Case: multiple chains
• Tertiary: describes the fold of a protein chain/subunit
• Quaternary: the arrangement of two or more protein subunits
Ribbon diagram
of a homodimer
of two protein
chains
N
C
Tertiary structure describes the
fold of each domain in a subunit
and their arrangement
(single chain)
Two domains
Two chains
N
C
Quaternary structure
describes the subunit
arrangement
(multiple chains)
Hierarchy of protein structure
Four domains
One chain
Four domains
Four chains
Src kinase: composed of four domains
Example of tertiary structure
neuraminidase: composed of four domains
Example of quaternary structure
Figure 3-10 Molecular Biology of the Cell (© Garland Science 2008)
Figure 3-21 Molecular Biology of the Cell (© Garland Science 2008)
Hierarchy of protein structure
N
C
Four domains
One chain
Four domains
Four chains
Src kinase: composed of four domains
Example of tertiary structure
neuraminidase: composed of four domains
Example of quaternary structure
Figure 3-10 Molecular Biology of the Cell (© Garland Science 2008)
Figure 3-21 Molecular Biology of the Cell (© Garland Science 2008)
Module 2: Protein structure
Video 11: Domains,
folds and modules
•
Associate Professor Terry Mulhern
96
What is Expected of You?
Learning Outcomes:-
(1) Explain the difference between the terms domain
fold and module.
What is a Domain?
Primary structure:
Secondary structure:
Tertiary structure:
amino acid sequence
arrangement of sequence into elements (α and β)
arrangement of elements into one or more domain
A single protein chain can consist of one or more domains.
• Although many proteins consist of a single polypeptide chain
they can be divided into separate domains.
• A domain is a region within the native tertiary structure for
which evidence can be provided of an existence independent
of the rest of the protein (fold independently)
One chain
Two domains
Hierarchy of Protein Structure
1. Primary structure: amino acid sequence
2. Secondary structure: elements, α-helix, β-sheet, turns
(simple motifs α−α, β−β, β−α−β –supersecondary structure)
3. Tertiary structure: folds, modules, domains, arrangement.
Nter-EEWYFGKITRRESERLLLNAENPRGTFLVRES -Cter
3
β−α−β
β1
α
β2
2
SH2
3
What is a Fold?
G6E
G6
G6E
XT
Src SH2
XR6 ute
A protein fold is defined by the arrangement of secondary structure elements relative to each other in space.
https://www.goauto.com.au/futuremodels/ford/falcon/range/ford-previews-2012-falconfacelift/2011-09-15/12513.html
Phosphotyrosine recognition domains: the typical, the atypical and the versatile.
Kaneko T1, Joshi R, Feller SM, Li SS. Cell Commun Signal. 2012 Nov 7;10(1):32. doi: 10.1186/1478811X-10-32.
What is a Module?
New proteins have evolved by
Gene duplication and domain shuffling
EGF
Protease
Kringle
Ca2+ binding
• Can attribute a function to individual folds: catalytic, lipid-binding, peptidebinding, DNA-binding, fibronectin-binding, transmembrane, etc.
• Many proteins are modular: have one or more repeating fold within their
overall structure – many transcription factors have three or more zinc fingers.
Hierarchy of protein structure
• Tertiary structure: the fold of a single protein subunit.
• Protein subunits can consist of more than one domain:
considered to be a discrete unit of protein folding, and can
be viewed independent of the rest of the protein subunit.
• Over half of domain structures can be broken down into
three repeating supersecondary structural elements: ααhairpin, ββ-hairpin, βαβ-element.
• There are 30,000 proteins (genes) →100,000 domains but
only 3,000 folds (prediction). ~2,500 different folds are
known so far.
• Soon, we should be able to predict the fold or identify the
domains of most proteins…
https://www.heraldsun.com.au/news/victoria/worst-traffic-times-melbourne-uber-driver-study-shows-tullamarine-freeway-the-most-frustrating/newsstory/f8fc66cb75efbe87f6c238d482c026fb
https://en.wikipedia.org/wiki/Road_train
https://www.youtube.com/watch?v=0iFkKRh5kcM
Module 2: Protein structure
Video 12: Protein
evolution
•
Associate Professor Terry Mulhern
105
What is Expected of You?
Learning Outcomes:1.
2.
3.
4.
5.
Define domain in terms of structure and evolution.
Describe the genetic processes that create proteins with different functional
properties using existing domain structures.
Explain what is meant by the statement that protein sequences can be optimally
aligned.
Explain the link between sequence identity, ancestry, and structural similarity.
Define homologue, orthologue and paralogue.
New proteins with new functions are been made by mixing domains and
mutating existing domains
New proteins with new properties and
functions can be generated by:
1. Intragenic mutation: point mutations,
insertions and deletions
2. Gene duplication: whole or part of a
genome is duplicated
3. DNA segment shuffle: two or more
existing genes can be broken (*) and
recombined
4. Gene lateral transfer: one organism
acquires parts of the genome of
another.
* Think of the broken “fragment”
as a domain or string of domains
A Domain can be defined as an evolutionary unit rather than a
structural unit: a brief introduction to bioinformatics.
• New proteins have evolved by gene duplication and domain shuffling. This
implies domains can have a common protein ancestor.
• The individual domains are further subjected to point mutations, insertions and
deletions (change, gain and loss of function).
• No new domains (folds) are being created – only modified and readjusted – (we
think nature has finished exploring protein fold space).
Original gene
maintains
phenotype and
function
Original gene:
• Mutations that do not effect function retained.
New gene gives
redundancy
Time
Gene
duplication.
New gene:
• Mutations may change/lose/gain function;
• environmental pressure selects if advantageous.
Mutagenesis allows
sequence to drift
Sequences may now differ
substantially……how different and what
has happened to the structure?
Sequences of protein domains can be aligned on the
basis of sequence residue identity or similarity
• Protein sequences can be aligned based on identical and similar
residues.
• Identity means exactly the same residue (invariant)
• Similar means a change to a residue that is observed frequently or with
similar physical-chemical properties eg Ser to Thr, Val to Leu (conservative)
• Alignments can be improved by introducing gaps that account for residue
insertions and deletions.
Similar residues
Identical residues
Better alignment by
introducing gap
Aligning two sequences of EF-Tu (Elongation Factors) from two bacteria
based on sequence identity and introducing a single gap.
(see Figs 3-33 and 3-34 Lehninger 6ed)
How do the structures of proteins that are related by such evolutionary
events such as gene duplication, but have different sequences, compare?
Identical residue, 31% identity
• Each sequence “change” results in a small change to structure.
• Structure, however, changes much more slowly than sequence. This means that
for two proteins whose sequences show >25% identity they will have a similar
structure.
• In general they will have similar secondary and tertiary structures. Gaps between
two sequences will result in a loop insertion within the general structural fold.
Primary amino acid sequence of domains can be aligned.
A general observation is – if the two sequences show > 25% identity the
protein domains are considered to be homologues.
To say two protein domains are homologues has special meaning – it
implies they arose from an event such as gene duplication (so they share a
common protein domain ancestor) *and* that means “they have the same
fold”.
Homologues can be divided into two groups:
• Orthologue: homologous proteins that perform the same function in
different species eg horse and tuna trypsin
• Paralogue: homologous proteins that perform different but related
functions within one organism eg human trypsin, compared with human
thrombin
If sequence identity is low <<25%, and proteins are functionally different, it is hard
to draw conclusions. If we find the fold to be similar then they are likely to be
homologues.
An example of probable gene duplication: chymotrypsin and
thrombin, two homologous (paralogues) serine proteases
• Sequences show 38% sequence identity
• The structures are strikingly similar – small
differences –loop size (*), extra helical turn (#),
but same β-structure and same overall fold.
Common Ancestor
Gene
duplication,
mutagenesis
• Each has the same functionally essential residues
that are involved in catalysis
#
*
*
*
• Differ in substrate specificity, and so binding
pockets have modified for specific function.
*
• structure changes more slowly than sequence
• Different parts of the protein mutate at different
rates (conservation of functional residues)
Chymotrypsin,
digestion
Thrombin,
blood clotting
8gch
1abi
H57
S195
D102
Overlay of functional
residues; same three
dimensional structure.