Lecture Notes for

Chapter 5
Protein Function
Essential Biochemistry
Third Edition
Charlotte W. Pratt | Kathleen Cornely
1
+
Protein Function (non-catalytic)
Well look at the following classes of proteins in this
chapter:
Binding proteins (bind and release small molecules, e.g. O
2
)

Structural proteins (shape of cell, some movement possible)

Motor proteins (convert chemical energy into physical
movement)

Next two chapters
Catalytic proteins (enzymes)
2
+
Why focus on
myoglobin and hemoglobin?
Sickled red blood cell Healthy red blood cell
O
2
transport is critical for sustaining life
Hemoglobin mutation can possibly lead to disease
Characteristics about O
2
binding to myoglobin and
hemoglobin are observed in many areas of biochemistry
3
+
Oxygen-binding Proteins
Both Mb and Hb carry a heme prosthetic group
Mb is a monomer; Hb is a tetramer
4
Structure of Myoglobin
Structure of Hemoglobin*
One subunit
*Identity of helices is an educated guess
Heme
group
A
B
C
D
E
F
G
H
+
Oxygen-binding Core
5
Heme group is prophyrin ring
Grk porphura: shellfish that yielded purple dye
(purple)
Polar propionate groups are solvent exposed
Nonpolar vinyl groups are buried
Central Fe
2+
ion coordinated to 4 N atoms of
porphyrin ring
Synthesized porphyrin derivative
+
Heme prosthetic group in Mb:
Also coordinated to His F8 residue (helix F below
ring)
Residue His E7 (above ring) forms H-bond with O
2

in oxy form
Heme by itself not a good O
2
carrier (Fe
2+
gets
oxidized to Fe
3+
)
In protected cleft in Mb & Hb
Oxygen-binding Core
6
His E7
His F8
O
2
proximal
distal
+
Binding of O
2
to Myoglobin
Lets define a constant to indicate equilibrium
binding of O
2



This is an association constant, but we could also express a
dissociation constant



This is a measure of the affinity of Mb for O
2
Once Mb binds O
2
, how tightly does it hold on?
As binding affinity , K

7
K
a
=
MbO
2
[ ]
Mb
[ ]
O
2
[ ]
K = K
d
=
Mb
[ ]
O
2
[ ]
MbO
2
[ ]
MbO
2
Mb + O
2
+
Binding of O
2
to Myoglobin

How can we measure how much O
2
is actually
bound?




Y = fractional saturation (O
2
-bound:total)
8

Y =
MbO
2
[ ]
Mb [ ] + MbO
2
[ ]
Mb bound to O
2
Total Mb
+
Binding of O
2
to Myoglobin

Now we want to combine our expressions for K and
Y to see how they relate
How does the affinity of Mb for O
2
(K) relate to the
degree to which it is saturated with O
2
(Y)
9

K =
Mb [ ] O
2
[ ]
MbO
2
[ ]

Y =
MbO
2
[ ]
Mb [ ] + MbO
2
[ ]
+
Binding of O
2
to Myoglobin

Rearrange expression for K and substitute:
10
MbO
2
[ ]
=
Mb
[ ]
O
2
[ ]
K
Y =
Mb
[ ]
O
2
[ ]
K
Mb
[ ]
+
Mb
[ ]
O
2
[ ]
K
=
Mb
[ ]
O
2
[ ]
K Mb
[ ]
+ Mb
[ ]
O
2
[ ]
=
O
2
[ ]
K + O
2
[ ]
=
pO
2
K + pO
2
pO
2
= partial pressure of O
2
The amount of O
2
bound to Mb is a
function of both the O
2
concentration
(pO
2
) and its affinity for O
2
(K)

K =
Mb [ ] O
2
[ ]
MbO
2
[ ]

Y =
MbO
2
[ ]
Mb [ ] + MbO
2
[ ]
+
Y versus pO
2
11
Y = Y
max
K = p
50
Why does Mb need to bind O
2
?
Mb delivers O
2
to mitochondria
Needed for reactions associated
with muscle activity
How does the fractional saturation of Mb (Y) change as pO
2
?
Hyperbolic plot
Binding increases rapidly until most molecules saturated
Value of K is where Mb is half saturated (2.8 torr)
Designated p
50
Y
max
+
Similarity of Mb and Hb
Hb is very similar in tertiary structure to Mb
Similar function (binding O
2
)
Homologous proteins
12
+
Similarity of Mb and Hb
Not that similar in primary structure (18% identical)
>1 sequence of amino acids can do the proper job of folding
Invariant residues: essential for function
Conservative substitutions: similar amino acids (Leu vs. Ile)
Variable: variety of residues possible
13
Identical in Hb &
Identical in Mb & Hb&
Invariant in all Mb & Hb
+
Binding of O
2
to Hb
14
O
2
binds to Hb differently than it does to Mb
Reaches same saturation point (Y
max
)
Curve is sigmoidal not hyperbolic
p50 (K) values very different
26 torr for Hb vs. 2.8 torr for Mb
Low K value high affinity


Hb
Mb
Y
max

+
Binding of O
2
to Hb
15
O
2
binds to Hb differently than it does to Mb
Affinity different at low vs. high pO
2
(slopes different)
At low pO
2
, O
2
reluctant to bind Hb
As pO
2
increases, more likely to bind


Hb
Mb
Y
max

+
Binding of O
2
to Hb
16
O
2
binds to Hb differently than it does to Mb
Affinity different at low vs. high pO
2
(slopes different)
This means binding is cooperative
4
th
O
2
bound with 100 times the affinity (more readily) as 1
st

Example of allostery
Grk: allos (other) + stereos (space)
Binding at one site affects affinity of other sites

Hb
Mb
+
Binding of O
2
to Hb
17
How would this be beneficial?
Bind in lungs, where pO
2
is high
Low affinity in tissues, releases O
2
there
This is the job of Hb transport O
2
from lungs to tissues
Mb affinity high so it takes up the released O
2



Hb
Mb
+
Conformational Shift in Hb
18
Deoxy is blue
Oxy is purple
O
2
In deoxy form:
Heme slightly bowed down (not planar or flat)
His F8 further from ring
Deoxy Hb Oxy Hb
Fe
2+
O
2
http://www.cryst.bbk.ac.uk/PPS95/course/10_i
nteractions/haemoglobin.html
F8
F8
E7
E7
+
Conformational Shift in Hb
19
Deoxy is blue
Oxy is purple
O
2
In oxy form:
O
2
binds, which pulls Fe
2+
into ring now
planar
H-bond forms with His E7
Pulls His F8 toward heme
This pulls F helix up by 1 (like moving 6
table by 2)
This changes quaternary conformation of
protein
Coordinated effort several things
happening at the same time
Deoxy Hb Oxy Hb
Fe
2+
O
2
http://www.cryst.bbk.ac.uk/PPS95/course/10_i
nteractions/haemoglobin.html
F8
F8
E7
E7
+
Conformational Shift in Hb
2 different responses at different levels of O
2

suggests 2 different states of Hb
1 for low pO
2
and 1 for high pO
2

Tense (T) state: conformation not favorable for O
2
binding
Relaxed (R) state: conformation now favorable for O
2
binding
20
T (tense)
state
R (relaxed)
state
Dimer of -
dimers
+
Conformational Shift in Hb
21
T (tense)
state
R (relaxed)
state
Fe
2+
becomes more planar in Oxy Hb
Fits better in heme ring
Has effect on subunit interface
This shift makes it easier to bind the next molecule of O
2
His on chain shifts between 2 Thr residues of chain in Oxy Hb
+
Bohr Effect and O
2
Transport
What is happening biochemically when you breathe?
From
Metabolism
+ H
2
O
22
+
Regulating O
2
Binding to Hb
Bohr effect
pH affects O
2
binding
HbH
+
+ O
2
HbO
2
+ H
+
Binding of O
2
lowers pK values of some residues
Donate H
+
more readily
As pH (i.e. [H
+
]), O
2
binding favored (equilibrium shifts right)
As pH (i.e. [H
+
]), O
2
release favored (equilibrium shifts left)
23
+
Regulating O
2
Binding to Hb
Bohr effect
What happens in tissues?
They use O
2
in respiration and produce CO
2
CO
2
diffuses into red blood cells (RBCs have Hb)
Carbonic anhydrase converts to bicarbonate and produces H
+


Low pH induces Hb to release O
2

Net effect: release O
2
in tissues, RBCs takes up CO
2
(Hb binds some)

24
CO
2
+ H
2
O HCO
3
+ H
+
From
Metabolism
+
Regulating O
2
Binding to Hb
Bohr effect
What happens in lungs?
Hb binds more O
2
in lungs releases H
+
Combines with bicarbonate (in red blood cell) to form
CO
2

CO
2
released from red blood cell and breathed out
Net effect: bind O
2
in lungs, release CO
2
25
CO
2
+ H
2
O HCO
3
+ H
+
Expel from
lungs
+
Effect of 2,3-bisphosphoglycerate (BPG)
Binds to Hb in deoxy (T) state and stabilizes it
Binding cavity too small in oxy (R) state
Favors O
2
release (not binding)
Regulating O
2
Binding to Hb
26
+ Regulating O
2
Binding to Hb
27
How O
2
gets to fetus
Mom has normal Hb binds BPG well
Hb in T state decreased O
2
affinity
Fetus has modified Hb binds BPG less
well Hb in R state increased O
2
affinity
Still sigmoidal!
O
2
passes from mother to fetus
Adaptation at high altitudes
Body produces more BPG
Lower affinity for O
2
means more
available in tissues
+
Structural Proteins
Cytoskeleton proteins
Distributed differently in cell
Microfilaments (~70)
Actin
Intermediate filaments (~100)
Keratin
Collagen
Microtubules (~240)
Tubulin

28
+
Two forms
Globular protein with ATP binding site: G-actin
Monomeric
Filamentous actin: F-actin
Double chain of subunits
(-) end has ATP site
(+) at opposite end
Actin Microfilament
29
ATP
G-actin
F-actin
(+)
(-)
(-)
(+)
+
Two forms
Monomers added together to form filament
Addition more rapid at (+) end
Driven by ATP hydrolysis
Catalyzed by F-actin (not monomer)
Most recently added actin bound to ATP
Actin Microfilament
30
+
ATP Hydrolysis
Major way the cell has to release energy
stored in chemical bond
Input energy to form bond
Releases energy when broken
Phosphate is a high energy bond
Takes a lot of energy to hold 2 phosphates
together repulsion from like (-) charges
Breaking that bond releases a lot of energy
Major energy currency in cell
Can spend it in many ways
Diffusible send it where you need to spend it

31
+
Actin Dynamics
Polymerization of actin is reversible
Grows and shrinks over time
Treadmilling: when addition = subtraction of subunits
Assembly and disassembly regulated
Can be used to extend and retract cellular processes
One method of cellular motility
32
Leading
edge
Trailing
edge
+
Microtubules
Built from dimers of tubulin
Forms protofilament (like actin)
Formed from dimers not monomers
Forms hollow cylinder, rather than single
filament
More rigid
Think about bicycle frames
33
+
Microtubules
Formation of microtubules
,tubulin
Each monomer binds GTP nucleotide
GTP blocked in but solvent-exposed
in
Formation of microtubule causes
hydrolysis of GTP in but not in
34
Tubulin dimer
GTP


+ Microtubules
Protofilaments (13) line up side by side to form tube
Dimers can add at both ends
(+) side faster
Disassembly faster at (+) end also
Used to construct cilia and flagella
Larger structural components
Used to align and separate pairs of chromosomes during mitosis
35
+
Intermediate Filaments
Exclusively structural proteins
Keratin
Basic unit is dimer of coiled helices (coiled coil)
7-residue repeat: #1 & #4 residues nonpolar
Line up along one side of helix
Hydrophobic van der Waals interactions hold them
together
36
+
Keratin
Two dimers associate to form
tetramers, two tetramers to form
octamer, etc.
Like winding several strands
together to form strong rope
Part of hair (-keratin) and nails (-
keratin) and skin
Intermediate Filaments
37
Skin cells
Dead epidermal cells
Mostly keratin
16-32 polypeptides
+
Collagen
38
Trimeric molecule
Part of animal bones and tendons
Holds cells together (French: glue)
Supports bodys weight
Most abundant protein in most vertebrates
Every third residue is Gly
30% of rest is Pro and Hyp
(hydroxyproline)
Triplets of Gly-Pro-Hyp most common
Forms narrow, left-handed helix

+
Collagen
39
Polypeptides wind around each other to form
right-handed triple helix
Gly residues in center of triple helix
No other residue would fit!
Triple helix stabilized by H-bonds
Via peptide bonds of Gly in adjacent helices
Peptide bonds of other residues interact with H
2
O
Also H-bonds between Hyp residues
+
Collagen
After secreted from cell that synthesized them,
proteases trim them
Align side-to-side and end-to-end to form large
fibers
Fibers have strong cross-links through oxidation of
Lys chains
CH
2
-NH
3
+
(C=O)H
40
Covalent bond of 2 oxidized Lys
+
Motor Proteins
41
Myosin and Kinesin
Basic structure the same
Mode of action different
Different functions in the cell
+
Motor Proteins
Myosin
2 polypeptide chains that form 2
heads and a long tail
Head has binding site for actin and
for ATP
2 small polypeptides (light chains)
wound around each neck of the
protein (4 total in dimer)
2 heads act independently
42
Myosin
Light
chain
Light
chain
+
Myosin Structure
Myosin tails associate to
form thick filament
Heads sticking out
Heads (multiple) bind to
actin thin filaments (part of
muscle fiber)
43
Rod of
myosin tails
Heads projecting out
+
Myosin Mechanism
44
45
Myosin Mechanism
*
*
46
Myosin Mechanism
+
Myosin heads does not remain bound to ATP/ADP
and filament at the same time
Power stroke at end of sequence
47
Myosin Mechanism
+
Kinesin
Microtubule-associated motor protein
Similar structure to myosin
Shorter neck
Light chains at other end of polypeptides
Light chains (two) bind some vesicle, with purpose of
moving it along a microtubule (2 light chains per
dimer)
Moves by stepping instead of grabbing
ATP hydrolysis drives the lever
48
+
Kinesin Mechanism
49
Head bound to ATP and
tubulin at same time
ATP affinity for tubulin
50
Kinesin Mechanism
51
Kinesin Mechanism
+
Kinesin Mechanism
Both myosin and kinesin are part of the P-loop
(phosphate-binding loop) superfamily of NTPases
(hydrolyze or digest Nucleotide TriPhosphate)
52
This is processive
One head always bound
Cargo remains attached and
keeps moving along
+
Kinesin Mechanism
How many light chains present in dimer as compared to myosin
dimer?
Does ATP binding increase or decrease binding affinity for
tubulin?
Both myosin and kinesin are part of the P-loop superfamily of
NTPases (hydrolyze or digest Nucleotide TriPhosphate)
53
+
Function of Kinesin
Motor proteins like kinesin
move cargo between the cell
body and the axon ends of
neurons
54
Neurons
Cell body
Axons