Download Chapter 7 and Protein Examples

Chapter 7 and Protein Examples
1) Ricin: Example of a multiple domain protein
The seed from the castor bean plant, Ricinus communis, are poisonous to people,
animals, and insects. One of the main toxic proteins is Ricin. Perhaps just 1 mg of ricin
can kill an adult (die from severe dehydration and a decrease in blood pressure). It is said
that just one castor bean seed can kill a child.
→Ricin is a heterodimeric type 2 RIP (ribosome inactivating protein). The ribosome
inactivating enzyme (32 kDa), also known as the A chain, is linked through a disulfide
bond to the galactose/N-acetylgalactosamine-binding lectin (34 kDa), also known as
chain B.
Type 1
RIP
wheat
barley
Type 2
RIP
S
S
lectin
Castor bean
On the 3-D ribbon structure of ricin above, the upper right half is the A chain, and the
lower left half is the B chain. The B chain is shaped like a barbell and will bind two
molecules of galactose at the ends. The A chain has 8 a-helices and 8 β sheets.
→The B chain allows the protein to bind to sugars in the outer membranes of cells. The
entire protein can then be endocytosed into the cytosol where the disulfide bond is
broken. The free A chain then catalyzes the depurinatio n of a specific adenine of the 28S
rRNA, halting protein synthesis.
Myoglobin:
Page 398 in text
→ Facilitates O2 transport in muscle.
→ O has a low solubility in water (10-4 M)
→ Myoglobin increases the effective solubility of O2 to boost O2 diffusion rate from
capillary to tissues.
→ Aquatic mammals have 10- fold greater myoglobin concentration-(used to store O2 )
Myoglobin is a single polypeptide with one heme group and hence one O2 binding site.
It is highly a-helical with 8 a helices, and it contains 1 heme group in a hydrophobic
pocket between the E and F helices (figure 12.14).
Heme group:
Heme is a protoporphyrin IX with an iron(II) atom in its center figure 12.12). The iron
atom can form six coordinating ligand bonds. The iron is coordinated to 4 nitrogen
groups within the heme structure The fifth coordinate bond is to a nitrogen of a
distal His residue within the protein. O2 forms a sixth coordinate bond, and is bonded
between the iron and another His residue on the protein (figure 12.15). The heme is
positioned within a hydrophobic pocket , where approximately 80 noncovalent
interactions are provided by about 18 amino acid residues to the heme. This pocket
protects the iron from being oxidized into iron (III).
→ Oxidation of the iron(II) to iron(III) yields metmyoglobin. Metmyoglobin will
not bind O2 . Some mutations result in a condition in which a person has a large
abundance of metmyoglobin and met hemoglobin. These people show a cyanosis (bluish
color of the skin). Mutating the distal His residue to Tyr results in this condition.
Oxygen Binding:
The binding of O2 to the heme group changes the structure of the heme group,
thus changing the absorption properties of the heme.
→oxygenated myoglobin- red (characteristic red color of muscles)
→deoxygenated myoglobin- blue
→ Fe+3 form- brown
→H+ and CO2 promote the dissociation of O2 from myoglobin (and hemoglobin).
Actively metabolizing tissues produce H+ and CO2 .
→NO and CO also have a high binding affinity for myoglobin (and hemoglobin)
Immunoglobulins (antibodies)
See page 393 in book.
Antibodies are immunoglobulin proteins secreted from B cells in the blood which
bind antigen in an immune response. Antibodies form a non-covalent association with
antigen, initiating a process by which the antigen can be eliminated from the body
(usually engulfed by macrophages). Each human can produce about 1x108 different
antibody structures (needed to handle all types of antigens).
All antibodies have a similar structure (figures 12.6 and 12.7). They have 4 polypeptide
chains held together by disulfide bonds.
→2 identical heavy chains (H), a variable amount of sugars are attached to the Hchain (2-12%)
→2 identical light chains (L)
→Have highly variable regions (V) at the N-termini which bind the antigen. The amino
acid sequence in the variable region gives a unique 3-D conformation (size, shape, and
non-covalent interactions) of the antibody that makes it specific to an antigen. The
antibody and antigen associate due to non-covalent interactions. Can bind identical
antigen molecules per antibody molecule.
→Have constant regions at the C-termini which determine the antibody class, bind
complement proteins, and are the site necessary to cross the placental membrane.
Antibody classes:
1) IgG- major immunoglobulin in plasma, best defense (but takes about 10 days),
promotes phagocytosis
2) IgM- found in plasma, first defense in plasma, promotes phagocytosis
3) IgA- found in all mucosal membranes, is deficient get recurrent infections of
sinuses and respiratory tract.
4) IgE- normal biological function unknown
5) IgD- normal biological function unknown may play a role in allergic reactions
and anaphylactic shock.
→All immunoglobulins have 6 domains. Each domain has similar folding: contain
antiparrallel β-strands which generate a motif known as an immunological fold. The
association of 2 folds creates a domain.
Immunizations:
An immunizing vaccine consists of killed bacterial cells, inactivated viruses,
killed parasites, a nonvirulent form of live bacterium, a denatured bacterial toxin, or a
recombinant protein. The antigens in these cause the activation and proliferation of B
cells so that they produce antibody toward the foreign antigen and also cause
differentiation of some B cells into memory B cells. The memory B cells act as a long
standing radar for the potentially virulent antigen. The binding of antigen to memory B
cells causes that cell to divide into antibody producing cells as well as new memory B
cells. This reduces the time required for antibody production on introduction of an
antigen and increases the concentration of antigen-specific antibody produced.
Example: Polio vaccine. Create memory B cells. If virus enters the body, antibodies are
quickly produced which help clear the virus from the body before it can reach the spinal
cord, where the virus causes polio.
Antibody column:
Used to separate a single biological molecule from a mixture. Antibodies specific
for the molecule of interest are covalently linked to a column. The mixture is then passed
through the column, and only the molecule of interest will bind to the antibodies in the
column. The column is then washed several times to remove all other material, and then
the molecule of interest is eluted from the column by denaturing the antibody.