§4 蛋白质功能Protein function(1)

liguofu
Structure of Porphyrin
Methene bridge
liguofu
Pyrrole ring
Oxygen can be bound to a heme (1)
None of the aa side chains in proteins is suited for reversible binding O2
3. Reversible interconversion of oxyhemoglobin and deoxyhemoglobin was revealed by using spectroscope (1860s).
4. Treatment of hemoglobin with acid gave a colorless albuminoid constituent (globin) and a red ironcontaining material (by 1870).
bindingsites total n[PLn ] n[P]
Байду номын сангаас
[L]n [L]n kd
liguofu
Quantitative description of interaction (2)
[L]n
[L]n kd
If kd is constant
liguofu
Quantitative description of interaction (3)
[L]n
[L]n kd
If kd is not constant
Kd
liguofu
Quantitative description of interaction (4)
[
[L]n L]n kd
[L]n 1 kd
log(
1
)
n
log[
L]
log
kd
liguofu
Quantitative description of interaction (5)
§4 Protein Function
§4.1 General features §4.2 Oxygen-binding proteins §4.3 Immunoglobulins §4.4 Muscle contraction
liguofu
§4.1 General features
1. Versatile in function 2. Being hard to study 3. Function via interaction
need Heme prosthetic group
Heme = Protoporphyrin IX + Fe2+
liguofu
more commonly
Transition metals, Fe & Cu, have a strong
tendency to bind O2
Oxygen can be bound to a heme (2)
Free Iron promotes the formation of highly reactive oxygen species such as hydroxyl radicals.
Coordinated N atoms help prevent the conversion of
Heme = Protoporphyrin IX + Fe2+ Fe2+ to Fe3+
liguofu
Hb has four subunits
146 residues
141 residues
liguofu
Mr = 64,500
Hb subunits are structurally
similar to myoglobin
D helix
subunit lacks
the short D helix
Also
In free heme molecules, reaction of oxygen at one of the two “open” coordination bonds of iron can result in irreversible conversion of Fe2+ to Fe3+
k
CO a
2000
k O2 a
or
k O2 d
2000
k
CO d
In myoglobin
liguofu
In myoglobin heme
kaCO
200
k
O2 a
or
k O2 d
200
k
CO d
In free heme
Protein structure affects ligands bind (2)
→ The interaction with other molecule (ligand) is reversible.
→ The interface between the binding site is complementary in structure, making such interaction highly specific.
→ Structure dynamicness of a protein is usually essential for such interactions.
liguofu
§4 Protein Function
§4.1 General features §4.2 Oxygen-binding proteins §4.3 Immunoglobulins §4.4 Muscle contraction
liguofu
143
conserved in all known globins
conserved
liguofu
82
27 positions are identical, ~18%
Interactions in the four subunits
19 residues
⋆Hydrophobic
interactions
“Breathing” :
Molecular motions
In myoglobin
kaCO
200
k O2 a
liguofu
The binding of O2 to heme in myoglobin
depends on its
“breathing”
Hb is the most-studied
and best-understood protein
Quantitative description of Mb binding O2
[O2 ]
[O2 ] kd
kd [O2 ]0.5
[O2 ]
pO2
[O2 ][O2 ]0.5 pO2 p50
liguofu
Protein structure affects ligands bind (1)
In free heme
⋆H bonds
⋆Salt bonds
30 residues
19 residues
liguofu
Ion pairs at the 1/2 and 2/1 interface (1)
Some ion pairs are not shown here
liguofu
This is called Tense state (deoxyHb)
Hill plot
liguofu
nH: hill coefficient
Hb undergoes a structural change on binding O2 (1)
Binding O2
Tense state is more stable and thus the predominant conformation of deoxyHb
liguofu
Quantitative description of interaction (1)
Protein + nLigand PLn
kassociation
[PLn ] [ P][ L]n
kdisassociation
[P][L]n [PLn ]
binding sites occupied n[PLn ]
liguofu
Val E11
Relaxed state is the predominant conformation of higher affinity to O2
– the first eukaryotic messenger (mRNA) to be isolated and subsequently sequenced.
– the first eukaryotic protein to be synthesized in a cell-free system in vitro;
Ion pairs at the 1/2 and 2/1 interface (2)
Ion pairs stabilize the T state of deoxyHb
liguofu
Quantitative description of Hb binding O2（1）
liguofu
Quantitative description of Hb binding O2（3）
– the second protein having its 3-D structure determined (1969).
liguofu
2. The “red coloring matter” (hemoglobin) of animal blood could be brought to crystallization, with crystal forms characteristic of their biological origins (1840s-1860s).
5. The structure of heme was elucidated to be a tetrapyrrol (porphyrin) derivative by chemical synthesis (Hans Fischer, 1929).
6. Similar tetrapyrrol derivatives are also used as prosthetic groups of other proteins (e.g., the cytochromes that function in biological oxidation and photosynthesis!
In heme-containing proteins, this reaction is prevented by sequestering the heme deep within a protein structure where acess to the two “open” coordination bonds is restricted. One of these two coordination bonds is occupied by a side-chain N of a His residue.
log( 1
)
n
log[
L]
log
kd
liguofu
The iron-porphyrin in hemoglobin accounts for the red color of blood, the copper-porphyrin in hemocyanin for blue color of blood, and the magnesium-porphyrin in chlorophyll is responsible for the green of plants.
1. Hemoglobin was
– the first protein to be crystallized (in 1849);
– the first to be associated with a specific physiological function (around 1875);
– one of the first proteins to have its molecular weight determined correctly (64,500);
liguofu
Mb has a single binding site for oxygen
His93 /F8
His64 /E7
Myoglobin:153 residues, 16700 Da
liguofu
Oxygen can be bound to a heme (3)
liguofu
NO CO