I have occasionally seen in almost dried blood, placed

1
Chapter 5 Protein Function
I have occasionally seen in almost dried blood,
placed between glass plates in a desiccator,
rectangular crystalline structures, which under
the microscope had sharp edges and were bright
red.
-Friedrich Ludwig Hunefeld, Der Chemismus

in der thierischen Organisation, 1840
(one of the
first observations of hemoglobin)
Since the proteins participate in one way or
another in all chemical processes in the living
organism, one may expect highly significant
information for biological chemistry from the
elucidation of their structure and their
transformations.
-Emil Fischer, article in Berichte der
deutschen
chemischen Gesellschaft zu Berlin, 1906
Knowing the 3-D structure of a protein is an
important part of understanding how the protein
functions
2
Ligand --- a molecule bound reversibly by a
protein Binding site --- the site on protein to
which a ligand binds Induced fit --- the
structure adaptation that occurs between
protein and ligand Substrate ---
the molecule acted upon by enzymes Catalytic/acti
ve site --- the substrate/ligand binding site
Noncatalytic functions of proteins
3
Reversible binding of a protein to a ligand
Oxygen-binding proteins
Heme
protoporphyrin
4
The structure of myoglobulin
a single binding site for O2 78 a helices
(8) His93 or HisF8 (the 8th residue in a
helix F) binds to heme Bends between a helices
5
Protein-ligand interactions can be described
quantitatively
P L PL
Ka PL/PL
Kd PL/PL
q (binding sites occupied)/(total binding
sites) PL/PL P L/(L 1/Ka)
q 0.5 L 1/Ka, or Kd
Graphical representation of ligand binding
6
P L PL Ka PL/PL
Dissociation constant, Kd PL/PL
q (binding sites occupied)/(total binding
sites) PL/PL P L/(L 1/Ka)
L/(L Kd) When L Kd q 0.5
(half saturation) L 9 Kd q
0.9 Kd the molar concentration of ligand at
which half of the available
ligand-binding sites are occupied Kd ,
affinity ( ? )
7
Table 5-1
8
When O2 binds to Mb
q L/(L Kd) O2/(O2 Kd)
O2/(O2 O20.5)
The concentration of a volatile substance in
solution is always proportional to its partial
pressure in the gas phase above the solution
q pO2/(pO2 P50)
9
Protein structure affects how ligands bind
1. Steric effects 2. Molecular motions/breathing
in the structure
(free heme)
Binding ability O2CO 1 20,000
1 200
10
Oxygen is transported in blood by hemoglobin (Hb)
In arterial blood, Hb 96 saturated In venous
blood, Hb 64 saturated
Mb has only one subunit, as an oxygen-storage
protein
Mb
11
Fig. 5-6 Comparison between Mb and Hbb
12
Fig. 5-7 Comparison of aa between whale Mb and
Hba, b
A-H helices
Only 27 aa identical
Pink conversed in all known globins
13
Fig. 5-8 Dominant interactions between Hb subunits
gt30 aa
19 aa (hydrophobic, H-bonds, affected strongly
upon O2 binding)
14
Hb undergoes a structural change on binding oxygen
O2
Fig. 5-10 The T(tense) R(relaxed)
transition
Max Perutz
15
Fig. 5-9 Some ion pairs that stabilize the T
state of deoxyHb
b1
a2
16
Fig. 5-11 Changes in conformation near heme on O2
binding
17
Hb binds oxygen cooperatively
pO2 4 (in tissues) vs. 13.3 (in lungs) kPa
(96)
Mb a single subunit protein Hb 4
subunits, an allosteric protein
(64)
Fig. 5-12 A sigmoid (cooperative) binding curve
18
Allosteric protein a protein in which the
binding of a ligand to
one site affects the binding properties of
another site on
the same protein
allos --- other stereos --- solid or
shape Homotropic interaction --- liagnd
modulator Heterotropic interaction --- ligand
modulator
O2 --- as both a normal ligand and an activating
homotropic modulator for Hb
19
Fig. 5-13 Structure changes in a multisubunit
protein undergoing cooperative binding to ligand.
20
Cooperative ligand binding can be described
quantitatively
P nL PLn Ka PLn/PLn
Dissociation constant, Kd PL n/PLn
Fig. 5-14
q (binding sites occupied)/(total binding
sites) Ln/(Ln Kd) q /(1- q)
Ln/Kd Logq /(1- q) n log L log Kd
(Hill equation, 1910) Logq /(1- q) n log
pO2 log P50 nH the Hill coefficient
(slope of Hill plot) When nH lt, , gt 1 ????
21
Two models suggest mechanisms for cooperative
binding
Fig. 5-15
Concerted (all-or-none), 1965
Sequential, 1966
22
Hemoglobin also transports H and CO2 (from the
tissues to the lungs and the kidneys)
CO2 H2O H HCO3-
Carbonic anhydrase in red blood cells
Lungs vs. Tissues
HHb O2 HbO2 H
His146 (His HC3)
O-
O
H
H
H
H
Bohr effect (Christian Bohr, 1904)
C - N- C- C-
C H2N- C- C-
R
R
O
O
O
O
Amino-terminal residue
Carbamino-terminal residue
Binding of H and CO2 to Hb favors a transition
to T state
23
K
R
24
O2 binding to Hb is regulated by
2,3-bisphosphoglycerate (BPG)
(heterotropic allosteric modulation)
HbBPG O2 HbO2 BPG
4
1
BPG during hypoxia
(highly abundant in erythrocytes) (8 mM at high
altitudes, 5 mM at sea level)
-
O O
-

O
C

C-O-P-O-
H-
-
H-C-H
O-
O
-O-PO
O-
Hypoxia--lowered oxygenation of peripheral
tissues
25
Positively charged aa
T
O2
T state
BPG is negatively charged
R
Fig. 5-18 Binding of BPG to deoxyHb
26
Sickle-cell anemia is a molecular disease of Hb
Glu6 mutates to Val6 in two b chains
27
Complementary interactions between proteins and
ligands The immune system and
immunoglobulins
MHC (major histocompatibility complex)
all vertebrate cells
macrophages, B cells
28
Over view of the immune response to a viral
infection
29
Structure of a human class I MHC protein
Recognized by T-cell receptor
30
The structure of immunoglobulin G (IgG)
31
Binding of IgG to an antigen
32
Induced fit in the binding of an antigen to IgG
Heavy chain
Light chain
Kd10-10M
33
The Ab-Ag interaction is the basis fro a variety
of important analytical procedures
Ployclonal vs. monoclonal Ab
ELISA (enzyme-linked immunosorbent assay)
34
Immunoblot assay (Western Blot)
35
Protein interactions modulated by chemical
energy Actin, myosin, and molecular
motors
Myosin
S1
36
The major components of muscle
37
Structure of skeletal muscle
relaxed
contracted
38
Muscle contraction
39
Molecular mechanism of muscle contraction
34 pN of forces, 510 nm movement/cycle