Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
27 Cards in this Set
- Front
- Back
|
Describe the 4º structure of antibodies.
|
It is stabilized by disulfide links
|
|
|
What do antibodies do?
|
Bind to specific antigens
|
|
|
What is the epitope of an antibody?
|
The specific area of antigen that the antibody recognizes
|
|
|
What is the difference between polyclonal and monoclonal antibodies?
|
Monoclonal: Can only recognize one epitope
Polyclonal: Can recognize a set of multiple epitopes |
|
|
Describe the structure of IgG.
|
• 4 polypeptide chains: 2 heavy and 2 light
• Linked by noncovalent and disulfide bonds • F ab region (top portion of y) if the antigen binding site: made up of variable and constant domains • Fc region: stem of y; Crystallizes readily; made up of constant domains |
|
|
How does IgG bind to an antigen?
|
Slight conformational changes upon binding to create optimal fit-> induced fit
|
|
|
How is binding specificity determined in antibodies?
|
• Determined by aa residues in variable domains of heavy and light chains
• The aa residues lining the AB site are hypervariable • Based on shape, location of charged, nonpolar, and H-bonding groups |
|
|
Discuss what a chromatography column does.
|
A selected Ab is covalently attached to a resin. When a mixture of proteins is added to the column, the Ab specifically binds the target protein and the rest wash through. Elution done with salt solution or other solution
• Really good for protein purification |
|
|
Discuss what ELISA is and what is is used for.
|
• Stands for Enzyme-linked Immunosorbent Assay
• Good for rapid screening and quantification of presence of antigen in sample |
|
|
Explain the method of ELISA.
|
1. Proteins in sample adsorbed to a surface
2. Surface washed with nonspecific protein to block proteins in subsequent steps from adsorbing 3. 1º Ab: Ab against the protein of interest 4. 2º Ab: Ab against 1º Ab: also linked to enzyme that catalyzes reaction that forms colored product 5. Substrate of 2ºAb-linked enzyme added 6. Color intensity (amt of product) is proportionate to amount of protein of interest present |
|
|
How is an immunoblot done?
|
1. Proteins that have already been separated by gel electrophoresis are transferred electrophoretically to nitrocellulose membrane
2. Membrane is blocked (like for ELISA) 3. Treated with 1º, 2º-with linked enzyme, substrate 4. Color product only forms along band that contains protein of interest |
|
|
What is an immunoblot good for?
|
Detection of minor component, and provides approximation of molecular weight
|
|
|
What is the function of myosin and actin?
|
Responsible for contraction of muscles
|
|
|
What is the structure of myosin?
|
6 subunits
• 2 heavy chains: formed l. handed coiled coil at C-terminus. N-terminus has globular domain where ATP is hydrolyzed • 4 light chains The myosin head group is responsible for the muscle contraction Aggregates to form thick filaments: rodlike, form the core of the contractile unit Tails associate to form long bipolar unit, globular domains project from either end |
|
|
What is the structure of actin?
|
G actin: monomeric; associated to form:
F actin: long polymer, filamentous Aggregate to form thin filaments along with Troponin and Tropomyosin |
|
|
Discuss the structure of muscle fibers.
|
• Each muscle fiber = 1 large, multinucleated cell that spans the length of the muscle
• 1 fiber = 1000 myofibrils • Myofibrils: consist of thin and thick filaments and other proteins • Sarcoplasmic reticulum: system of vesicles that surrounds each myofibril |
|
|
Explain the parts of a sarcomere.
|
• I band: only thin filaments
• A band: includes region of overlap of thick and thin filaments Z disk: bisects I band: perpendicular to it and is the anchor where thin filaments are attached M line: bisects A band |
|
|
Explain how a muscle contracts.
|
1. ATP binds to myosin, disrupting its interaction with actin, and Myosin releases the actin
2. ATP is hydrolyzed: causes conformational change in myosin that causes the head to change its orientation in relation to actin 3. P group released as myosin head attaches to actin filament 4. Pi release triggers the power stroke -> conformational change in myosin head that returns it to its original resting state. ADP released in the process |
|
|
How is muscle contraction regulated?
|
Tropomyosin is bound to the thin filament, where it blocks the attachment site for myosin head group
Nerve impulse releases Ca2+ from sarcoplasmic reticulum Troponin binds Ca2+, causes conformational change in Tropomyosin, causes the myosin-binding site to be exposed |
|
|
What are the 2 molecular functions of myosin?
|
1. Binding: actin-binding protein
2. Catalysis: ATPase |
|
|
Explain the limitations of O2 that necessitate a heme group to be used to transport it.
|
O2 is poorly soluble in aqueous solutions
Cant be carried to tissues if it is dissolved in blood Cant diffuse over long distances AA side chains can't reversibly bind it > Use transition metals: Iron and Copper But free iron binding to O2 creates free radicals that are harmful to DNA/macromolecules -> USE A HEME |
|
|
What is the structure of heme?
|
Organic ring: porphyrin
1 Fe2+ bound Has 6 binding sites: 4: N 1: proximal His 1: oxygen |
|
|
What is the structure of myoglobin?
|
Monomer: single polypeptide
153 aa, 1 heme attached 8 alpha helices (A-H) connected by bends |
|
|
What is the structure of hemoglobin?
|
4 subunits: with 1 heme in each
2 types of globin: 2 alpha chains and 2 beta chains 4º structure: features strong interactions between unlike subunits Strongest interfaces: a1B1 and a2B2 : involve over 30 residues Weakest: a2B1, a1B2: only 19 residues |
|
|
What are the 2 conformations of hemoglobin?
|
R: relaxed
T: tense O2 binds to both but has a higher affinity for R |
|
|
What happens when O2 binds to a hemoglobin subunit in the T state?
|
Causes it to change to R state and aB subunits slide past each other and rotate which narrows the pocked between B subunits
|
|
|
Is hemoglobin an allosteric protein?
|
Yes, binding of 1 O2 changes the affinities of any remaining unfilled binding sites
O2 is an activating homotropic modulator |
