Lecture 8: Protein Structure

Question 1

A molecule that binds to a protein is called?

Answer 1

A ligand

Question 2

A region in the protein where the ligand binds is called

Answer 2

The binding site

Question 3

What type of bonding interaction do ligands bind?

Answer 3

Noncovalent interactions

Question 4

What is Ka and Kd. Draw their equations based on a Protein (P) and Ligand (L)

Answer 4

k_a [P][L] = k_d [PL]

k_a = [PL] / [P][L] = 1/k_d

k_d = [P][L] / [PL]

Question 5

How can we directly measure [PL]

Answer 5

P_tot = [P] + [PL]

[PL] = P_tot [L] / K_d + [L]

Question 6

What it the equation for fraction of occupied binding sites?

Answer 6

Or x = [PL] / [PL] + [P]

Question 7

What is the gibbs free equatino with the dissociation constant

Answer 7

ΔG° = RT ln(k_d)

Question 8

What does a strong binding or weak binding look like with k_d

Answer 8

Strong binding: k_d < 10 nm

Weak binding: k_d > 10 um

The smaller the k_d the strong the bond

Question 9

What is the lock and key model?

Answer 9

Assumes that complementary surfaces of enzymes are preformed

Complementary in

1. Size
2. Shape
3. Charge
4. Hydrophobic/philic character

Question 10

What is induced fit?

Answer 10

Conformational changes may occur upon ligand binding to enzyme and ligand

• allows for tighter binding
• allows for high affinity for different ligands

Question 11

What is cooperativity?

Answer 11

As the change in properties of binding at one site as a function of whether or not another site is occupied

• Often caused by structural change from binding ligand on one site
• can make other sites stronger or weaker

Question 12

What is the Hill equation

Answer 12

n = Hill coefficient (the degree of cooperativity)

Question 13

What biological problems do we have with O2

Answer 13

• Proteins lack affinity for it
• Binding to some transitional metals would create free radical
• Fe3+ is very reactive

Question 14

What is the biological solution to O2

Answer 14

Capture oxygen molecule with heme that is protein bound

Question 15

Why is CO better at binding heme than O₂

Answer 15

because the carbon in CO has a filled lone electron pair that can be donated to the vacant d-orbitals on the Fe²⁺

Question 16

How does O2 binding affect the color or Fe

Answer 16

• Fe²⁺ without O₂ has intense soret band at 429 nm
• With O₂ shifts to 414 nm
• Via UV-Vis
• Deoxyhemoglobin appears purple
• oxyhemoglobin appears red

Question 17

For effective transport, affinity must vary with pO₂

Answer 17

Question 18

What is Tense state and Relaxed state

Answer 18

Tense state

• More interactions, more stable
• lower affinity for O₂

Relaxed State

• Fewer interactions, more flexible
• higher affinity for O2

Question 19

Does O2 cause a T → R change or R → T change?

Answer 19

O2 binding trigger a T → R conformational change

Involves breaking ion pairs between the a1-b2 interface

Question 20

What is the a1-b2 interface?

Answer 20

Question 21

What triggers the conformational change in hemoglobin?

Answer 21

By oxygen binding

Question 22

What effect does pH binding have on hemoglobin

Answer 22

• H⁺ binds to Hb and stabilizes the T state this protonates His146 which then forms a salt bridge with Asp94
• Leads to the release of O₂
• Increases K_d

Question 23

What is the Bohn effect

Answer 23

The effect pH has on the binding of O₂ to hemoglobin. This increases the efficiency of the O₂ transport.

Question 24

What is carbamate?

Answer 24

15-20% of CO2 is exported in the form of a carbamate on the amino terminal residue of each of the polypeptide subunits

It produces an H contributing to Bohr effect

Stabilizes T state

Question 25

How does 2,3 Bisphosphoglycerate regulate O₂? (know structure)

Answer 25

1. Produced as intermediate of glycolysis
2. Small negatively charged molecule, bind to the positively charged central cavity of Hb
3. Stabilizes the T state and allows for better release in Tissue and adaptation to changes in altitude (higher K_d)

Question 26

What mutation on hemoglobin occurs to form sickle cell

Answer 26

Glutamate-6 to valine

Question 27

Who are the key players for cellular immunity?

Answer 27

Targets own cells that have been infected

macrophages, killer T cells, and inflammatory T cells

Question 28

What is humoral fluid immune system

Answer 28

Targets extracellular pathogens

makes soluble antibodies

Keyplayers: B-lymphocytes and helper T cells

Question 29

How does an antibody look?

Answer 29

Two heavy chains and two light chains

Light: one constant and one variable domain

Heavy: Three constant and one variable domain

Variable Domain: make up the antigen-binding site (two per antibody) and are hypervariable.

Question 30

How do antigen bind?

Answer 30

Via induced fit

Question 31

How does ELISA work?

Answer 31