Topic 3 - Protein Structure (General Study Questions)

Question 1

Why do disulfide bonds in protein need to be reduced?

Answer 1

1. intramolecular disulfide bonds: They interfere with sequencing when trying to determine the primary (1o) protein structure
2. intermolecular bonds: means there are 2 different polypeptide samples present; the sample is not pure and must be purified from each other

Question 2

How do you reduce a disulfide bond?

Answer 2

1. DTT: dithiothreitol - a reductant that reduces disulfide bond by becoming a disulfide bond itself
2. When you do not want free thiols in solution - use TCEP
3. beta-mercaptoethanol: can also reduce; more commonly used when SDS gel will also be used

Question 3

What problems arise from reducing a disulfide bond & how do you solve them?

Answer 3

1. Reoxidation - interfere with sequencing
2. Reoxidation could also result in new non-native disulfide bonds - disulfide bonds that form where there was originally none
   Solution: alkylation - irreversible modification of cysteines. Use iodoacetamide to convert SH into a long compound so that it cannot reoxidize

Question 4

What do you use to cleave 1o structure polypeptide into smaller fragments for sequence determination?

Answer 4

Endopeptidases

Question 5

What does the endopeptidase Trypsin cleave after?

Answer 5

Positively-charged aa: Arginine & Lysine (AL/RK)

Question 6

What does the endopeptidase Chymotrypsin cleave after?

Answer 6

Bulky, hydrophobic aa: Phenylalanine, Tryptophan, Tyrosine (PTT/FWY)

Question 7

What does the endopeptidase Elastase cleave after?

Answer 7

Small, neutral aa: Glycine, Alanine, Valine, Serine

Question 8

What does the endopeptidase Cyanogen Bromide cleave after?

Answer 8

Methionine

Question 9

What method(s) can you use to determine the sequence of a polypeptide?

Answer 9

1. Edman degradation: sequentially determining N-terminal amino acids using Edman’s reagent, which will modify the N-terminus. It removes that aa and determines what it is, then removes the next aa…
2. Mass Spec: determine mass of long peptide, chop up that peptide, determine fragment sizes, determine order

Question 10

What are the steps to determine a polypeptide sequence?

Answer 10

1. Reduce disulfide bonds - they interfere with sequencing. Use CTT, TCEP, or beta-mercaptoethanol
2. Cleave into smaller fragments using endopeptidases
3. Determine sequence using Edman degradation (sequential) or Mass Spec
4. Generate overlapping fragments

Question 11

What is 2o protein structure?

Answer 11

Folding of polypeptide backbone

Question 12

What are the characteristics of the peptide bond?

Answer 12

1. resonsance of bond = partial double-bond characteristic
2. no free rotation/planar/no conformational changes
3. always trans configuration except for proline

Question 13

What are the constraints to 2o structure?

Answer 13

1. Planar peptide bond with R groups in trans

2. Rotation: bulky R groups, same charge on R groups, Proline (steric hindrance)

Question 14

What is a Ramachandran Plot?

Answer 14

A plot that allows you to see all theoretically possible and observed constraints

Question 15

What stabilizes 2o structure alpha helices?

Answer 15

1. Hydrogen bonds between backbone atoms
2. Attractive side-chain interactions between aa’s that are 3- 4 positions apart
3. Opposite charges
4. Aromatic amino acids: aromatic stacking contributes more interaction

Question 16

What destabilizes 2o structures alpha helices?

Answer 16

1. Proline (Helix breaker)
2. Aromatics next to each other: too bulky
3. Similar charges close together: repulsive = destabilizing
4. Glycine: too many will destabilize b/c they are flexible

Question 17

How do we measure secondary structure?

Answer 17

circular dichroism (CD) - Sample absorbs right- and left-circularly polarized light to a different extent

Question 18

What stabilizes tertiary & quaternary protein structure?

Answer 18

1. Interactions of amino acid side chains - sedcondary in determining/stabilizing structure
2. Hydrophobic forces - primary determinant, based on hydrophobic effect
3. Electrostatic Forces: Hydrogen bonding, dipole-dipole interactions, ion pairs
4. Covalent bonds
5. Ion pairs/Salt Bridges: more in 4o structure