Protein Function II 10

Question 1

Immunoglobulin G (an antibody)

Answer 1

• IgG consists of 2 heavy chains and 2 light chains
• stabilized by intra and inter-chain disulfide bonds
• organized into complement and 2 antigen binding regions
• has constant domains and variable domains (at the top)

Question 2

Immunochemical detection

Answer 2

• the antigen binding region of Fab consists of residues from both heavy and light chains (at the top)
• specific binding to the antigens epitope is primarily due to the complementary surface, Hbonds and electrostatic forces
• antibody specificity can be used to detect and quantify selected proteins and other molecules, either directly (e.g. ELISA) or following protein separation by electrophoresis (Western blotting)

Question 3

ELISA

Answer 3

• enzyme linked immunoabsorbent Assay
• uses multiple antibodies to specifically label and detect antigen on a micro tiger plate
• most commonly used lab diagnostic method for proteins and other molecular biomarkers

Question 4

Western blotting

Answer 4

-uses antibodies to specifically label and detect proteins following their separation, usually by SDS-PAGE

Question 5

Actin filaments (microfilaments)

Answer 5

• Fibrous (F) actin is a polymer of globular (G) actin monomers, with a diameter of 70A
• many functions: cortical skeleton underlying plasma membrane, and essential for cellular motility
• F-actin filament has polarity (has a positive end and a negative end), so it can reversible polymerize at both ends (faster at positive end)
• F-actin is organized into cables, meshes by over 100 different actin binding proteins

Question 6

F-actin assembly

Answer 6

• G-actin and F-actin are in equilibrium in most cells
• ATP bound to G-actin is hydrolysis to ADP after incorporation into F-actin; which is accompanies by a small conformational change
• polymerization is usually more rapid at + end
• capping proteins can block F-actin polymerization at + end while severing proteins break filaments
• fungal compounds stabilize or destabilize F-actin

Question 7

F-actin treadmilling

Answer 7

-hydrolysis of ATP also allows F-actin to treadmill, with net polymerization at + end and removal at - end

Question 8

Actin in cell motility

Answer 8

• stress fibres create contractile bundles (antiparallel)
• cell cortex forms gel-like network
• filopodium forms tight parallel bundles

Question 9

Actomyosin: a molecular machine

Answer 9

• drives muscle contraction
• can contract up to 40%
• uses ATP to induce conformational changes
• thin filament = actin
• thick filament = myosin
• myosin consists of a tail, head and neck, and are intertwined

Question 10

Actomysocin contraction cycle

Answer 10

1. The reaction sequence begins with a myosin head bound to an actin subunit of the think filament. ATP binding alters the configuration of the myosin head to that it releases actin
2. The rapid hydrolysis of ATP to ADP +Pi triggers a conformational change that rotates the myosin lever and increases the affinity of myosin for actin
3. Myosin binds to an actin subunit father along the thin filament
4. Binding to actin causes Pi and then ADP to be released. As these rxns products exit, the myosin lever returns to its original position causing the thin filaments to move relative to the thick filament (power stroke).
5. ATP replaces the lost ADP to repeat the reaction cycle
   - muscle is rigor (dead/stiff) if there is a lack of ATP, it becomes locked.

Question 11

Microtubules

Answer 11

• largest cytoskeletal element and are hollow for increased rigidity
• have structural and transport roles in the cell
• MTs provide straight and rigid tracks for movement of chromosomes and vesicles, and form cilia and flagella
• target of anti-cancer drugs

Question 12

Microtubule structure

Answer 12

• reversible aggregates of tubulin diner, forming hollow tubes
• 250A wide
• MTs are polar: often polymerize and depolymerize from same + end (regulated by GTP): split ends
• negative end is often anchored to an organizing centre (eg. Centrosome)

Question 13

Kinesins transport cargo along microtubules

Answer 13

1. ATP binding to leading head induces a conformational change in which the neck docks against the head. This movement swings the trailing head forward by 180* toward the + end of the microtubule. This is the force-generating step.
2. The new leading head quickly binds to a tubulin sun unit and releases its ADP. This step moves kinesins cargo forward along the protofilaments
3. In the trailing head, ATP is hydrolyzed to ADP + Pi. The Pi diffuses away, and the trailing head begins to detach from the microtubule
4. ATP binds to the leading head to repeat the reaction cycle.

Question 14

Intermediate filaments

Answer 14

• large heterogeneous family of insoluble fibrous proteins (100A in diameter)
• IFs provide mechanical strength and shape to cells
• lack polarity and nucleotide binding, but (de)polymerization may be regulated by phosphorylation
• examples include lamins underlying the nuclear membrane and keratin in epithelial cells
• dimers and tetramers coil together to form rope like structures.

Question 15

Coiled-coil structure of IFs

Answer 15

• fundamental unit of intermediate filaments is helical could
• strengthened by non-polar residues between helices

Question 16

Fibrous proteins: collagen

Answer 16

• most abundant protein in body (25% of total): twisted brain of 3 extended chasing (1,100A)
• forms strong cables and meshes in the extracellular matrix connective tissue and bone
• largely composed of Pro-Hyp-Gly repeats
• Diseases of collagen include scurvy (lack of VitC), Ehlers-Danlos (stretchy skin) and osteogenesis imperfect (brittle bone)

Question 17

Collagen triple helix

Answer 17

• Glycine at every 3rd residue is critical for triple helix to form (only flexible reside compared to Pro, Hyp)
• interchain hydrogen bonds are also strengthened by hydroxolysine and hydroxyproline residues

Question 18

Fibrous proteins: elastin

Answer 18

• deformable protein found in elastic tissues (lungs, blood vessels)
• Lys cross-linking connects chains of monomers
• cleaved in alveolar wall by elastase, which is inhibited by a1-antitrypsin
• decreased a1-AT activity in emphysema can be cause by genetic mutations or environmental factors