Major component of connective tissue of vertebrates.
Consists of three left-handed helical chains coiled around each other in a right-handed
supercoil.
Each helix has 3 amino acids per turn and a pitch of 0.94 nm _ more extended than an ahelix.
Stability of the collagen helix is achieved via interchain hydrogen bonds.
Helical regions consist of the amino acids –Gly-X-Y, where X is usually proline and Y is
usually hydroxyproline.
For each –Gly-X-Y triplet, one hydrogen bond forms between the amide hydrogen atom of
glycine in one chain and the carbonyl oxygen of an adjacent chain.
There are no intrachain hydrogen bonds.
Hydroxyproline and hydroxylysine are made from proline and lysine after the protein has
been synthesized, i.e. an enzyme does the hydroxylation.
In mammals, vitamin C is necessary for adequate hydroxylation.
People who suffer from scurvy lack sufficient amounts of vitamin C in their diet.
Develop skin lesions, fragile blood vessels (susceptible to bruising), loose teeth, and
bleeding gums.
Collagen triple helices are arranged in a staggered fashion to give rise to very strong fibers.
There are some covalent cross-links between the side chains of some lysine and
hydroxylysine residues to form Schiff bases between carbonyl groups and amines.
6
MYOGLOBIN AND HEMOGLOBIN
Vertebrates must supply and deliver a constant amount of oxygen to tissues for aerobic
respiration. This is done in two ways:
1) development of circulatory system that delivers oxygen to cells
2) use of oxygen-carrying molecules to overcome oxygen’s low solubility in water
e.g. myoglobin and hemoglobin
The ability of myoglobin or hemoglobin to bind oxygen depends upon a heme group
(prosthetic group).
Heme consists of an organic part (protoporphyrin) and iron atom.
Iron atom in center can form 6 bonds: 4 with nitrogens from protoporphyrin and 2 on
either side of plane.
Iron atom can be in ferrous (+2) or ferric (+3) state --> ferrohemoglobin and
ferromyoglobin and ferrihemoglobin and ferrimyoglobin. Only +2 state can bind oxygen
Myoglobin structure determined with x-ray crystallography in mid 1950’s.
Molecule has several important features:
1) extremely compact
2) 75% of structure in a-helix (8 helices, named A, B, C, ...H).
3) 4 of helices are terminated by proline residue
4) main-chain peptide groups are planar
5) little empty space inside molecule; interior consists almost entirely of nonpolar
residues; amino acids that are amphipathic oriented so that hydrophilic portions
face exterior; only polar amino acids in interior are 2 histidines, which are part of
binding site.
Heme group located in crevice in myoglobin molecule.
Iron atom is bonded to histidine in F8 (histidine); the oxygen-binding site on iron is located
on other side of heme plane (E7).
Binding of oxygen to heme must occur in a bent, end-on orientation.
If only a small portion of the protein binds oxygen, why have the rest of the protein?
Heme exposed to oxygen by itself rapidly oxidizes to +3, which cannot bind oxygen.
Heme is much less susceptible to oxidation because not only allows heme to bind oxygen, but
it is a reversible process.
Carbon monoxide is a poison because it combines with ferromyoglobin and ferrohemoglobin
to block oxygen transport.
CO’s binding affinity is about 200x stronger than that for oxygen.
If allow CO to interact with isolated iron porphyrins, the iron, carbon, and oxygen atoms are
in a linear array.
If allow CO to interact with myoglobin or hemoglobin, CO axis is bent, as in oxygen binding
because of steric hinderance from His E7 --> greatly weakens the interaction of CO
with the heme.
7
Biological significance?
CO is produced within cells in the breakdown of heme --> about 1% of binding sites on
hemoglobin and myoglobin are blocked by CO.
If affinity was close to that of isolated iron porphyrins --> massive poisoning.
Bottom line: function of a prosthetic group is modulated by its polypeptide environment.
Hemoglobin consists of 4 polypeptide chains, 2 of one type, 2 of another (M
2
,M
2
), held
together by noncovalent bonds.
Each polypeptide contains a heme group and oxygen binding site.
Embryos and fetuses have zeta chains (M) and epsilon (M) chains; zeta is replaced by
alpha (M), epsilon chains are replaced with gamma (M), then beta (M) chains.
The three-dimensional structures of myoglobin and and chains of hemoglobins are very
similar --> myoglobin resembles a chains of hemoglobin.
Odd because amino acid sequence is not very similar --> different amino acid sequences can
specify similar 3-D structures.
Those amino acids found to be invariant (do not change) are those directly bonded to heme
iron or hold helices together.
The nonpolar character of interior of molecule is conserved --> important in binding heme
group and stabilizing 3-D structure of each subunit.
Hemoglobin is more intricate than myoglobin.
1) transports protons, carbon dioxide, and oxygen
2) is an allosteric protein
3) binding of oxygen to hemoglobin is cooperative
4) affinity of hemoglobin for oxygen is pH dependent; same true for CO
2
5) hemoglobin also regulated by 2,3-bisphosphoglycerate (BPG)
If look at oxygen dissociation curves for myoglobin and hemoglobin, find many differences:
1) saturation of myoglobin is higher at all oxygen pressures than hemoglobin -->
myoglobin has higher affinity for oxygen than does hemoglobin P
50 for myoglobin is 1
torr; P
50
for hemoglobin is 26 torr
2) oxygen dissociation curve of myoglobin is hyperbolic; that of hemoglobin is sigmoidal -
-> binding of oxygen to hemoglobin is cooperative (seen in Hill plot)
Biological significance of cooperativity?
Enables hemoglobin to deliver nearly twice as much oxygen under typical physiological
conditions as it would if binding sites were independent
8
Effects of pH on Oxygen Binding
Decreases in pH shift oxygen dissociation curve to the right --> hemoglobin affinity for
oxygen is decreased.
Effects of CO
2
on Oxygen Bindin
Increases in carbon dioxide concentration lower hemoglobin’s affinity.
Both of these actually promote the release of oxygen from oxyhemoglobin.
All of these effects are known as the Bohr effect.
Effect of BPG
Lowers oxygen affinity of hemoglobin by a factor of 26 --> hemoglobin unloads more of its
oxygen at the tissue level.
BPG works by binding to deoxyhemoglobin, but not oxyhemoglobin.
Differences between fetal and adult hemoglobin:
Hemoglobin F (
2
2
) vs. hemoglobin A (
2
2
)
Fetal hemoglobin has higher affinity for oxygen than does hemoglobin A --> optimizes
transfer of oxygen from maternal to fetal circulation.
Also, hemoglobin F binds BPG less strongly than does hemoglobin A --> higher oxygen
affinity, but in the absence of BPG, fetal hemoglobin actually has lower affinity for
oxygen than does adult hemoglobin.
Structural Basis of Allosteric Effects
The allosteric properties of hemoglobin arise from interactions between its subunits.
The functional unit of hemoglobin is a tetramer with 2 alpha and 2 beta chains.
The structures of oxyhemoglobin and deoxyhemoglobin are very different.
1) oxygenated molecule more compact
2) binding of oxygen to hemoglobin results in a large structural change at two of the
four contact points (
1
2
and
2
1
)
3) the 11 pair rotates relative to other pair of protein chains
The 12 contact region is designed to act as a switch between two alternative structures.
All mutations in the interface diminish oxygen binding; mutations elsewhere do not.
Oxyhemoglobin is R form; deoxyhemoglobin is T form (lower affinity)
In deoxyhemoglobin the iron atom is out of porphyrin plane toward proximal histidine (F8) --
> heme group is domed-shaped toward His F8.
Binding of oxygen to iron atom moves iron atom into porphyrin plane --> heme becomes more
planar.
Proximal histidine is pulled along with iron atom and becomes less tilted --> shifts F helix ---
> --> transmitted to subunit interfaces, where they break interchain salt links ---> R
form
9
How does BPG lower oxygen affinity of hemoglobin?
Only one molecule of BPG is bound --> binds to symmetry axis of hemoglobin molecule
in central cavity.
This binding site contains 8 positively charged residues: amino group, His 2, Lys 82, His 143
of each beta chain.
BPG has 4 negative charges.
When oxygen binds to hemoglobin, the shift in conformation causes the central cavity to
become too small --> BPG is expelled.
BPG stabilizes deoxyhemoglobin structure by cross-linking M chains --> shift equilibrium
toward T form.
How does CO
2
lower oxygen affinity of hemoglobin?
CO
2
is transported as bicarbonate in RBCs
CO
2
+ H
2
0 HCO
3
-
+ H
+
Much of the H
+
is taken up by deoxyhemoglobin in the Bohr effect
Remainder is bound to hemoglobin as carbamate
R-NH
2
+ CO
2
R-NH-C-O
-
+ H
+
Carbamate forms salt bridges that stabilize T form --> lowers affinity
Uptake of H+ helps buffer pH of metabolically active cells, but also must raise the pKs of
some of the amino acids
The only a.a. affected is His 146, which acquires a greater affinity for H
+
because local
environment (location of Asp 94) becomes more negatively charged.
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