Chapter 5: Function of Globular Protein

Question 1

What are 5 functions of Globular Proteins? Give examples of each.

Answer 1

Storage of ions and molecules (myoglobin, ferritin), Transport of ions and molecules (hemoglobin, serotonin transporter), Defense against
pathogens (antibodies, cytokines), Muscle contraction (actin, myosin), Biological Catalysis (chymotrypsin, lysozyme).

Question 2

How many Amino Acids are in each turn of an Alpha Helix?

Answer 2

3.6

Question 3

What is a Ligand? What forces does it bind with and why?

Answer 3

A small molecule that binds to something else. Typically binds via non-covalent factors which enables the interactions to be transient
(Hydrophobic Effect, Hydrogen Bonding, Van Der Waals).

Question 4

In terms of drugs, what do rate constants/kinetics tell us?

Answer 4

These are used to determine the affinity of a drug – good vs. bad drug.

Question 5

What units are used to describe the Dissociation Constant (Kd)? What is Kd equated to? What type/size of Kd is favorable?

Answer 5

M for Molarity (mols/L). It is equated to the ligand concentration that leads to half-maximal saturation of the protein binding site. Small
Kd is favorable - because it means we have a large numerator, which shows poor binding since it means lots of proteins and ligands are
free in solution.

Question 6

What level of affinity is the binding by enzymes? Why?

Answer 6

Low affinity binding - because it needs to release its product.

Question 7

What Kd degree is considered ‘strong binding’? Weak binding?

Answer 7

Strong is Kd less than 10^-9 (nanomolar) and Weak is Kd greater than 10^-6 (micromolar).

Question 8

Describe the Law of Mass Action in relations to the formation of protein-ligand complexes.

Answer 8

At high ligand concentration, we will get formation of a complex regardless of Kd value.

Question 9

What is the Langmuir Isotherm Equation?

What equation is used to normalize the data?

Answer 9

[PL] = [P]t [L]t / Kd + [L]t
Non-linear regression fitting graph describes a square hyperbola that is typical of saturable binding and provides an estimate of Kd.
Theta = [L]t / Kd + [L]t

Question 10

What is Fractional Occupancy?

Answer 10

Y/Ymax. Basically divide all concentration values by Ymax.

Question 11

What 3 assumptions must be satisfied for the Langmuir Isotherm Equation to be true?

Answer 11

The binding is at equilibrium, the binding is reversible, and the free ligand concentration is basically the same as the total ligand
concentration because the amount of protein in the assay is so small.

Question 12

What could affect Kd values in a reaction solution? How/Why?

Answer 12

pH. Ligand or protein are affected by protonation.

Question 13

Describe the Induced Fit Theory. What makes it a driving force for co-operativity?

Answer 13

It is the explanation of ligand binding that conformational changes occur upon ligand binding. This induced fit allows for tighter binding of
the ligand. Induced fit can also increase the affinity of the protein for a second ligand, which makes it a driving force for co-operativity.

Question 14

Why is Myoglobin needed in the body? How does it work?

Answer 14

Myoglobin is a protein needed to store oxygen for metabolism because protein side-chains lack affinity for O2 and some transition metals
bind O2 well but generate free radicals if free in solution. Myoglobin captures the O2 molecule with a Heme moiety that is sequestered
inside a protein.

Question 15

What makes up the backbone of heme? What do the conjugated double bonds help with?

Answer 15

Porphyrin ring. Conjugated double bonds allow the molecule to absorb light easily (chromophore).

Question 16

What 4 substituents does the Fe in Myoglobin attach to?

Answer 16

Histidine residue, oxygen, and 2 sides of the Porphyrin ring.

Question 17

How can the binding of Oxygen to Myoglobin be detected?

What colors do deoxy and oxymyoglobin appear as to the naked eye?

Answer 17

The Heme group is a strong chromophore that absorbs in the ultraviolet and visible range. Form without Oxygen (Ferrous) has intense
soret bands at 429nm and form with Oxygen has soret band at 414nm.

Deoxymyoglobin appears as purple and Oxymyoglobin appears as red.

Question 18

What is Hemoglobin?

Answer 18

A tetramer of 2 subunits (alpha 2 beta 2) that are very similar to myoglobin (in their secondary and tertiary structures.

Question 19

What 3 Amino Acids are involved in the Hemoglobin Triad? What are the interactions between them?

Answer 19

Lysine, Histidine, and Aspartate.
Electrostatic Interactions (Carbonyl terminus of Histidine + Amino terminus of Lysine as well as Carbonyl terminus of Aspartate + Amino
terminus of Histidine)

Question 20

Describe the T state of Hemoglobin.

Answer 20

(Tense) T-State = Lower pH (7.2) = Tighter Triad = Lower affinity for oxygen (Deoxyhemoglobin subunits are more stable in this state).
In this state, Oxygen binding triggers a conformation change to the R-State as the system straightens out as oxygen binds more.

Question 21

Describe the R state of Hemoglobin.

Answer 21

(Relaxed) R-State = Higher pH (7.6) = Looser Triad = Higher affinity for oxygen (Oxyhemoglobin subunits are more stable in this state).

Question 22

What happens to which ion pairs during the conformational change from the T to R state of Hemoglobin?

Answer 22

Breaking ion pairs between the alpha-1- beta-2 interface.

Question 23

What is the general effect of pH on Oxygen binding? Where do we find blood with higher pH? Lower pH?

Answer 23

Oxygen binds well at higher pH and is released at lower pH. Higher pH (7.6) found in lungs while lower pH (7.2) found in capillaries of
metabolic tissues.

Question 24

Describe the Bohr Effect. What is a major contributor to the Bohr Effect and how?

Answer 24

Bohr Effect is the shift of the hemoglobin binding curve to the left at higher pH (more O2 affinity) and to the right at lower pH (less O2
affinity). A major contributor to the Bohr Effect is the Histidine residue on the triad which causes it to favor the T-state at lower pH
because here, it is protonated (lower pH = acidic environments) due to its interaction with Aspartate. When deprotonated, Histidine
favors the R-state

Question 25

What is the effect of CO2 on Hemoglobin’s oxygen affinity?

Answer 25

CO2 is produced by metabolic tissues and must be exported. Some as bicarb dissolved in blood and others as Carbamate on the amino
terminal residues of each Hemoglobin Polypeptide Subunit. Because there is so much CO2, the reaction between CO2 and the amino
terminal residue occurs, forming a C-N bond and kicking off a proton that can bind to Hemoglobin (His) (solution becomes more acidic).
pH decreases, and the T state is favored, which decreases Oxygen affinity.

Question 26

What does BPG contain? Where does it come from? What is the effect of BPG on Hemoglobin’s affinity for Oxygen? What state, T or R, is
stabilized by BPG?

Answer 26

BPG contains lots of phosphoryl groups and is naturally present in erythrocytes. It causes the hemoglobin binding curve to be more
sigmoidal - decreases Hemoglobin’s affinity for Oxygen. It is an allosteric effector. BPG stabilizes the T-state.