Week 3: Globular Proteins

Question 1

What is a globular protein?

Answer 1

polypeptide chains are folded into compact structures unlike fibrous proteins. The polypeptide chain is often folded into 1+ kind of secondary structure. These regions must be folded on each other - tertiary structure.

Question 2

What is the diversity of globular proteins? What is an example?

Answer 2

Diversity: sequence, function, fold. Enzymes, provide chemical energy, transport of oxygen for aerobic organisms, selective import molecules etc.

Question 3

What is responsible for the fold of globular proteins?

Answer 3

The fold is way secondary structure elements are connected - packing - tertiary structure

Question 4

What is the most important part of globular protein for defining function?

Answer 4

Outer surface

Question 5

What are the 3 common motifs in protein structure?

Answer 5

1. Dominant secondary structural motifs (mainly alpha helical, mainly beta sheet, both)
2. Proteins have multiple domains (compact, locally folded region of tertiary structure, 150-250 amino acids - interconnected by polypeptide strand that runs through whole moelcule). Diff ones have diff functions.
3. Domains may be composed of repeating secondary structure motifs.

Question 6

What is a tandem repeat?

Answer 6

contiguous arrangement of multiple copies of a repeating structural unit in linear sequence on protein chain.

Question 7

What are the 4 basic folding patterns of globular proteins?

Answer 7

1. built from packing of alpha-helices
2. constructed on beta sheet framework
3. have both helices and sheets
4. have little helix or sheet structure.

Question 8

What determines secondary and tertiary structure?

Answer 8

Amino acid sequence

Question 9

What is denaturation? How does it occur? Is it reversible?

Answer 9

natural structure of protein is lost along with many functional properties. Occurs by increase in temp, change in pH, addition of chaotropic molecules ie. urea. Denaturation is reversible by lowering temp, restoring pH or removing chaotrope. A protein then self-assembles into its functional conformation - needs no information.

Question 10

Is the folding of globular proteins favorable under physiological conditions?

Answer 10

Yes, it is thermodynamically favorable, free energy change is negative

Question 11

What is the equation for free energy change?

Answer 11

DeltaG = DeltaH - TDeltaS

Question 12

What happens if free energy is below zero, above zero or zero?

Answer 12

If free energy decreases Delta G < 0, it is spontaneous, if free energy increases Delta G > 0 - not spontaneous. For protein that spontaneously folds under physiological conditions, Delta G (folding) < 0

Question 13

What does a negative Delta H mean? How does this contribute to free energy change (delta G)?

Answer 13

Heat of reaction, if negative, heat released. The major source for negative Delta H is energetically favorable interactions between groups within folded molecule, contributes to a favorable Delta G

Question 14

What is delta S? What does it mean if it is less than zero?

Answer 14

Entropy is measure of randomness in system. Entropy gain is favorable and loss in unfavorable.

Involves decrease in randomness and decrease in entropy Delta S <0. This is conformational entropy of folding: random coil (higher entropy) → folded protein (lower entropy). The conformational entropy change works against folding. Folding results in loss of conformational entropy Delta S (conformation) for protein folding is negative - favors unfolded state

Question 15

What are charge-charge interactions? What happens if pH is too high or low? How does this impact delta H?

Answer 15

Occur between charged side-chains - electrostatic attractive force between - salt bridges - ionic bonds are broken if protein is taken to pH values high or low enough that side chains lose charge. The mutual repulsion between pairs of similarly charged groups further contribute to this. Each interaction between opposite charges contributes to favorable Delta H for folding.

Question 16

How does internal hydrogen bonds contribute to delta H?

Answer 16

If amide protons or carbonyls in polypeptide backbone are not involved in secondary structure formation, they are potential candidates for interaction with side-chain groups. Contributes to favorable delta H for folding.

Question 17

What is the change in enthalpy for folding (deltaH U->F) dominated by? How do these interactions affect delta H

Answer 17

differences in noncovalent bonding interactions between unfolded and folded states - where unfolded state is characterized by noncovalent interactions between extended protein chain and solvent water molecules and folded state includes many fewer interactions with solvent and more intramolecular interactions instead. The only new covalent bonds that form upon folding are disulfide bonds. Each interaction contributes to favourable delta H for folding.

Question 18

What is the hydrophobic effect? How does this affect entropy?

Answer 18

The ordering of clathrates corresponds to a loss of randomness in solvent - entropy of solvent is decreased. When polypeptide chain is unfolded, these residues are in contact with water and cause ordering of surrounding water into clathrates. When chain folds, the hydrophobic residues become buried within and water molecules that were ordered around are now released from clathrates, gaining freedom of motion - randomness of solvent is increased. The overall change in entropy is sum of the change in conformational entropy for polypeptide chain and change in entropy of solvent water molecules:
The burial of hydrophobic SA in solvent-inaccessible core stabilizes the folded state by making Delta S{U ->F} more positive. This is hydrophobic effect.

Question 19

Summarize what the stability of folded structure of globular proteins depends on. See image

Answer 19

1. conformational entropy change Delta S (protein) which favors the unfolded state
2. Enthalpy change, Delta H (associated with changes in noncovalent bonding interaction), which generally favors the folded state
3. The favorable entropy change of the solvent due to release of water from clathrates, Delta S{solvent} which occurs when solvent-exposed hydrophobic groups become buried in molecule.

Question 20

How can the native functional conformations be perturbed?

Answer 20

changes in intracellular conditions. For many proteins, there is a well-established relationship between activity of certain proteins and its native conformation - allowing its activity to be regulated. Some of these regulations are reversible and others are irreversible.

Question 21

What is the role of disulfide bonds in protein stability?

Answer 21

Once folding has occurred, the 3D structure can be further stabilized by formation of disulfide bonds between cysteine residues.

Question 22

What are cofactors/prosthetic groups?

Answer 22

The folded conformation can be stabilized by binding of metal ion (ie. zinc finger proteins in DNA) or by binding to a cofactor or prosthetic group - usually occur in small domains (< 100 residues) where there may not be enough noncovalent interactions.

Question 23

What are zinc finger domains in DNA?

Answer 23

Zinc finger domains are maintained by binding zinc to side chains of two histidine and 2 cysteine residues.

Question 24

What is an apoprotein? A holoprotein?

Answer 24

When the ion or cofactor is absent the “stripped” protein is called an apoprotein and when ion or cofactor is bound, the resulting complex is a holoprotein - its formation stabilizes the active conformation because favorable noncovalent interactions are formed between apoprotein and bound ion or prosthetic group. If remove the metal, the fold is weakened or lost.

Question 25

What interactions are involved in quaternary structure? What does it result in?

Answer 25

The interactions between these polypeptide chains include - salt bridges, hydrogen bonding, van der Waals forces and disulfide bonding, along with hydrophobic effect.

The Quaternary structure leads to increased stability of folded state, assembly of large structures, in enzymes it is major form of activity control.