Biochem Exam #2

Local _____ structures fold first. In these, ______ play an important role. Longer range interactions follow. In these, _______ plays a significant role. Then, the process continues until the entire polypeptide folds.

secondary ; ionic interactions ; hydrophobic effect

Free-energy funnel: Unfolded states have a (high/low) degree of conformational entropy, meaning (high/low) free energy. Therefore, the protein is (unstable/stable) with a (high/low) ΔG.

high ; high ; unstable ; high

Final folded/native state of a protein:
(high/low) ΔG?
(high/low) entropy?

low ; low

What are the 2 types of chaperone proteins?

• Hsp70

• Chaperonins

What does the Hsp70 chaperone protein do?

Binds to the hydrophobic regions on nascent (not fully synthesized) proteins and binds to misfolded nascent peptides to correct their misfolding.

What do the Chaperonins chaperone proteins do?

Fix misfolded mature proteins by fully encapsulating the protein to repair its misfolding.

What diseases can misfolded proteins lead to?

• Alzheimer’s

• Parkinson’s

• Prion diseases (mad cow and Creutzfeldt-Jakob disease)

The healthy functional proteins tend to be (water-soluble/insoluble). The harmful functional proteins tend to be (water-soluble/insoluble).

water-soluble ; insoluble

Why do harmful functional proteins tend to be insoluble in water?

Because misfolding flips the protein inside out, exposing its hydrophobic interior.

A harmful functional protein, specifically a _____, infects any protein it comes in contact with in a cascading effect, which further causes the progression of the disease.

prion

What is the first protein to have its structure determined via X-ray crystallography?

Myoglobin

Myoglobin has a ______ structure. It has _____ subunit(s).

tertiary ; one (1 subunit → binds to 1 O₂)

Both myoglobin and hemoglobin reversibly bind to _____. Therefore, they are both perfect models for studying _____.

O₂ ; protein function

What protein is a tetramer of myoglobin-like subunits?

Hemoglobin (4 subunits → each subunit binds to 1 O₂)

What organic molecule is a protein-bound prosthetic group found in both myoglobin and hemoglobin?

Heme

What structure does heme have?

A protoporphyrin ring structure with a bound Fe²⁺ atom

• 4 nitrogens

• Fe³⁺ does not bind to O2

How many coordination (covalent) bonds does Iron (Fe²⁺) form?

6

• In a regular covalent bond, each atom brings one electron. In a coordination bond, the other molecule brings both electrons (bc Fe2+ is electrophilic)

Iron (Fe²⁺) forms ____ coordination bonds to nitrogen atoms in the flat porphyrin ring (heme) and ____ perpendicular coordination bonds to the porphyrin.

4 ; 2

Putting it together, hemoglobin has ____ subunits, each with a heme porphyrin ring (with a Fe²⁺ in the center). Since Fe²⁺ forms ____ coordination bonds, with 1 of those reserved for binding to O₂, hemoglobin as a whole reversibly binds to a total of ____ O₂ molecules.

4 ; 6 ; 4

Of the 2 perpendicular bonds that Fe²⁺ forms, one of them is reserved for binding O₂. The other is occupied by a ______ of a ______ (proximal/distal) His residue.

side-chain nitrogen ; highly conserved ; proximal

Differences between Proximal vs Distal Histidine

	Proximal Histidine (His F8)	Distal Histidine (His E7)
Position	Directly below/closer to the heme iron	Above/farther from the heme iron on the opposite side
Iron Interaction	Forms a direct covalent coordination bond with Fe²⁺	Does not bond directly to the iron atom (helps O2 bind to Fe2+ in heme)
Interaction	Anchors the heme group inside the protein pocket	Forms hydrogen bonds with bound O₂ to stabilize it

Globins are a family of ______ proteins. Most function in O₂ _____ or _____.

oxygen-binding ; transport ; storage

Globins have a highly conserved _______ structure (1 subunit), with ____ α-helical segments connected by bends (globin ____).

tertiary ; 8 ; fold

Hemoglobin has a ______ structure.

quaternary (think about it: made up of 4 subunits - myoglobin 4x)

What is a Globin motif(fold)?

helices connected by loops or turns

What are the 4 types of globins found in humans and mammals?

• Myoglobin (monomeric)

• Hemoglobin (tetrameric)

• Neuroglobin (monomeric)

• Cytoglobin (monomeric)

What is the difference between myoglobin and hemoglobin in terms of function?

• Myoglobin: facilitates O₂ diffusion in muscle tissue

• Hemoglobin: responsible for O₂ transport in the bloodstream

How many residues does myoglobin have and how many molecules of heme? How many helices does myoglobin have?

• 153 residues

• 1 molecule of heme

• 8 α-helices (labeled A through H)

______ is the link between heme and myoglobin.

Proximal His (His F8)

Proximal His (His F8) is the ______ residue from the amino acid terminal end of myoglobin.

93rd

In myoglobin, His F8 is the ____ residue in the F helix specifically.

8th

Protein (P) - Ligand (L) interactions are _______.

reversible

In P-L interactions, what is Y?

Fraction of binding sites that are bound

• never reaches 1

What is K_D?

Dissociation constant (in mol/L or M) → equilibrium constant for the release of ligand

• K_A = 1/K_D, therefore we use K_D bc it is easier to use M than M^-1

[L] (x-axis) at which half of the available ligand-binding sites are occupied, Y = 0.5 (y-axis), corresponds to _____.

K_D

• Y = 0.5 → K_D on graph

lower K_D = (higher/lower) ligand binding affinity

higher

In the graph showing the binding of O₂ to Myoglobin, Y = 0.5 (y-axis) corresponds to _____ on the pO₂ (kPa) x-axis.

P50

• 
  

  

  P₅₀ is the oxygen equivalent of K_D (same concept, just diff name bc it is a gas)

Myoglobin’s Distal His E7 (increases/decreases) heme’s affinity for O₂.

increases

Distal His E7 acts as a ______ in myoglobin by ______ to control ligand access to the heme pocket.

gate ; rotating

The "pocket" refers to the heme pocket (also called the ligand-binding pocket), which is a deep, ______ cavity inside the myoglobin or hemoglobin protein structure where the oxygen-binding heme group

hydrophobic

Adult hemoglobin has 2 globin types: ______ and ______. How many residues are in each?

• Two α chains (141 residues each)

• Two β chains (146 residues each)

Important note: Alpha/beta chains are just names of the hemoglobin subunits, and both hemoglobin chains are all α-helix with no β-sheets.

Proteins with very different amino acid sequences can fold into _____ 3D shape.

the same

• Similar structure can arise from different sequences, and it’s the structure that determines function. Different sequence doesn’t automatically mean different function, as long as the fold is preserved.

What are the 2 conformations of hemoglobin?

R-state and T-state

R-state (relaxed) = O₂ has a higher/lower affinity for hemoglobin

higher

T-state (tense) = O₂ has a higher/lower affinity for hemoglobin

lower

Deoxyhemoglobin is the oxygen-free form of hemoglobin. What conformation is Deoxyhemoglobin in?

T-state

What is the T-state stabilized by?

A greater number of ion pairs (ionic bonds within and between its subunits), many of which lie in the α₁β₂ and α₂β₁ interface

• ionic interaction between Lys and His

T = tense = more/less ion pairs = tightly locked. R = relaxed = more/less ion pairs = loose and ready for oxygen.

more ; less

During the T → R transition, there is no longer an ionic interaction between ____ and ____. The ____ and ____ subunits begin to rotate: Pocket is _____ and _____ rotates toward the center but is no longer in ion pair → structural change → functional change (can bind to O₂ now).

Lys ; His ; α ; β ; narrowed ; His C3

Is the pocket more narrow in the T or R state?

R state

In the T-state, how is the porphyrin ring of heme affected?

The porphyrin ring is puckered (nonplanar) because Fe²⁺ is pulled out of the plane of the ring toward the proximal histidine (His F8).

In the R-state, how is the porphyrin ring of heme affected?

The porphyrin ring becomes planar when O₂ binds, causing a shift in the position of the proximal histidine (His F8) and other residues in helix F.

• This conformational change helps stabilize the R-state, which has a higher affinity for O₂

Cooperative binding in hemoglobin is where oxygen binding to one site increases/decreases the affinity of the remaining sites, making it easier/harder for more oxygen to attach (domino effect).

increases ; easier

Hemoglobin has a ______ binding curve for oxygen.

hybrid sigmoid (S-shaped)

What is an allosteric protein? What is an example?

Binding of a ligand to one site affects the binding properties of another site on the same protein

• Hemoglobin

What are modulators? What is an example?

Ligands that bind to an allosteric protein to induce a conformational change

• Oxygen

What are the 2 types of modulators? Explain each.

• Homotropic: ligand and modulator are identical

• Heterotropic: ligand and modulator are not the same molecule

Which molecule is a homotropic modulator of oxygen binding to hemoglobin?

Oxygen

• Why? Bc modulator = ligand

What are the 2 models that suggest mechanisms for cooperative binding? Briefly define each.

• MWC model/concerted model (all or nothing): all four subunits change conformation simultaneously → ligand binds more tightly to the R-state

• Sequential model (domino effect): subunits change conformation one at a time and each change increases the O₂ affinity of neighboring subunits, progressively favoring the R-state.

  • In the sequential model, hemoglobin can have a mixture of T-like and R-like subunits, whereas the MWC model does not allow this.

Explain how hemoglobin is a hybrid between the 2 models that suggest mechanisms for cooperative binding.

1. The initial binding of O₂ to hemoglobin begins with the sequential model because when O₂ binds to the first subunit, only one subunit conformational change occurs (still in T-state)

2. When two or more O₂ bind to the protein → R-state

3. It ends with the concerted model after 2 or more O₂ bind to the 2nd/3rd subunit where they undergo a concerted conformational change → R-state