Flashcards in Topic 3 - Protein Structure (General Study Questions) Deck (18):
Why do disulfide bonds in protein need to be reduced?
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
How do you reduce a disulfide bond?
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
What problems arise from reducing a disulfide bond & how do you solve them?
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
What do you use to cleave 1o structure polypeptide into smaller fragments for sequence determination?
What does the endopeptidase Trypsin cleave after?
Positively-charged aa: Arginine & Lysine (AL/RK)
What does the endopeptidase Chymotrypsin cleave after?
Bulky, hydrophobic aa: Phenylalanine, Tryptophan, Tyrosine (PTT/FWY)
What does the endopeptidase Elastase cleave after?
Small, neutral aa: Glycine, Alanine, Valine, Serine
What does the endopeptidase Cyanogen Bromide cleave after?
What method(s) can you use to determine the sequence of a polypeptide?
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
What are the steps to determine a polypeptide sequence?
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
What is 2o protein structure?
Folding of polypeptide backbone
What are the characteristics of the peptide bond?
1. resonsance of bond = partial double-bond characteristic
2. no free rotation/planar/no conformational changes
3. always trans configuration except for proline
What are the constraints to 2o structure?
1. Planar peptide bond with R groups in trans
2. Rotation: bulky R groups, same charge on R groups, Proline (steric hindrance)
What is a Ramachandran Plot?
A plot that allows you to see all theoretically possible and observed constraints
What stabilizes 2o structure alpha helices?
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
What destabilizes 2o structures alpha helices?
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
How do we measure secondary structure?
circular dichroism (CD) - Sample absorbs right- and left-circularly polarized light to a different extent