Chapter 5: Protein Function

Dr. Clower
Chem 4202

Functions of Proteins

Protein Function
Very specific biological function
Varies based on structure
Fibrous proteins
Enzymes
Transport across membranes

Common theme: how proteins bind to interact

with other molecules
Involves reversible binding with ligands (or substrates)
in binding site (or active site)
Can have multiple binding sites in one protein
Binding may involve change in conformation (induced fit)
Induced fit may cause change in other parts of the
protein (e.g. other subunits)
Interactions between ligands and proteins are regulated

Model Proteins
Myoglobin and Hemoglobin
Oxygen-binding proteins
Transport
Increase solubility of O2 in aqueous
environment

First 3D structures determined

Myoglobin
Mb
Transport protein
Primarily in muscle
tissue
Structure
Small, globular
153 residues
8 -helices (A-H) connected
by bends (AB, etc.)
Contains one heme group
Prosthetic group
Binds in hydrophobic pocket

Heme
Found in O2 transport proteins and
electron-transfer proteins
(cytochromes)
Heterocyclic porphyrin ring system
Conjugated
Flat
Binds divalent ion
e.g. Fe2+

Located deep in protein structure

to prevent oxidation of ion
Between E and F helices
Held in place by hydrophobic
interactions (Phe, Val)

Heme
Iron has 6 coordination
bonds
4 to pyrrole groups
1 to His residue
Proximal His
F8; His93

1 to O2 or other small
molecule (CO, NO)

Binding of Oxygen
Equilibrium
Ka = association constant
Not the same as acid dissociation constant
Measure of affinity of L for P
Higher Ka = higher affinity

Kd = dissociation constant
Lower Kd = higher affinity

Fractional saturation ()
Fraction of ligand-binding sites occupied by L

Kd = [L] at which
binding sites
occupied
Protein is halfsaturated
Lower Kd = smaller
[L] required due to
stronger binding
between L and P

Reversible Binding of O2 to Mb
Equilibrium expression
Kd expression
Substitute [O2] = pO2
Partial pressure
Easier to measure

Substitute Kd = [O2]0.5
Concentration at which sites occupied
P50

Expression for

Plot of vs. pO2 for Mb

Low pO2 = low binding

Increase pO2 = increased binding until saturated

Steric Factors

O2 vs. CO binding
Free heme vs. Mb
heme
Distal His = E7 (His

64

Hemoglobin
Hb
Closely related to Mb
More O2 transport
Multiple subunits
Multiple binding sites
Responsiveness to changes in pO2

Hb Structure
Spherical
Tetrameric protein
Quaternary structure =
= 141 residues
= 146 residues
Both and similar to Mb
Structure, not sequence

Amino Acid Sequences of Mb and Hb

Hb Structure
Dimer of protomers
Rotational symmetry

Hb structure
and units attract at interfaces
1 1 and 2 2
35 residues

2 1 and 1 2
19 residues

Typically hydrophobic
Also electrostatic and H-bonding
1 2 and 1 2 little or no interaction
Separated by solvent channel

Each subunit binds a heme

Between E and F helices
Heme binds O2
Structure changes when O2 binds

Deoxyhemoglobin
Very little O2
affinity
Some electrostatic
interactions

T State

R State

Oxyhemoglobin
Higher affinity of O2
dimer rotates ~15
2 1 and 1 2 contacts
shift
chains closer together
Some ion pairs broken

O2 Binding to Hb
pO2 higher in lungs than in tissue
O2 needs to bind, then release
This will not happen with Mb
hyperbolic curve; animation

Hb transitions T to R state as more O2

binds
Cooperative interaction between
binding sites
Binding to one site affects binding to
the other sites
One O2 molecule binding increases O2
affinity of other sites
Allosteric protein

Plot of vs. pO2 for Hb

Hb described by
sigmoidal curve
At low pO2, sites
compete for first O2
ligand and weak
binding in T state
Slope increases
quickly due to
increased affinity of
other sites (T R)
Becomes saturated

Hill Equation
Describes sigmoidal curve
Expression for
n = number of binding sites

Hill plot
nH = slope = Hill coefficient
Measure of degree of cooperativity (interaction
between binding sites)
Solving Hill equation shows that the 4 th ligand
binds with 100x greated affinity than 1 st ligand

nH = 1
Hyperbola (like Mb)
Ligand binding is not
cooperative

nH > 1
Positively cooperative
(like Hb)
Upper limit = n (4 for Hb;
typical nH ~2.8-3.0; upper
limit never reached)

nH < 1
Negatively cooperative
Reduce affinity when first
ligand binds

Hill Plot

Explanation for Cooperative Interaction

Why does ligand affinity increase?
How does one heme affect the others?
Not through electronic mechanism
Hemes too far apart (25 37 )

Due to change in structure upon

oxygenation
Perutz mechanism
Change from T state conformation to R state
conformation

Perutz Mechanism
Very fast
1.

Fe(II) in T state site above heme

Fe(II) binds to O2
Fe(II) pulled down into heme (R state)

2.

Fe(II) pulls down His F8

F helix tilts

Animation

Perutz Mechanism
3.

Shift of tertiary structure causes shift of quaternary

structure (rotate)
2 1 and 1 2 interface residues realign

4.

C-terminal residues break ionic interactions which

stabilize T state

As R state forms from T state, it adopts ideal

conformation for next O 2 binding

All binding sites are altered, not just the one binding the
O2

Bohr Effect
Conformational change

will be accompanied by
change in IFs
Change in charge

Also, H+ and O2 compete

for binding to Hb
Relate pH to affinity
Bohr effect
O2 affinity increases as pH
increases
Animation (YO2 = )

Regulation of O2 Binding
O2 affinity affected by other molecules which can bind to the protein
CO2
D-2,3-bisphosphoglycerate (BPG)
Binds to T state (central cavity)
Does not bind to R state
Keeps Hb in deoxy form
Decreases O2 affinity
Allow ligand to be released

Animation

CO2H

OPO32-

OPO32-

Chapter 5 Problems
1-6