Lecture 5

Question 1

options for protein sequencing (3)

Answer 1

1. Sanger method for direct protein sequencing
2. Sequencing from DNA sequence databases
3. Mass Spectrometry

Question 2

Sanger protein sequencing: 3ish steps

Answer 2

1. Break disulfide bonds
2. tag amino terminus
   - FDNB attacks, then strong acid
   3a. cut protein into smaller pieces
   3b. cut protein into small pieces in different spots than 3a so have pieces that overlap with 3a

Question 3

cleaving disulfide bonds

Answer 3

performic acid or dithiothreitol

Question 4

label n-terminus

Answer 4

FDNB then strong acid. N-terminus stays attacked to the FDNB. all peptide bonds broken (hydrolyzed)

Question 5

Cutting large proteins

Answer 5

1. Trypsin
   - cleaves carboxyl side of (+) R groups
2. Chymotripsin
   - cleaves carboxyl side of Aromatic R groups
3. CNBr
   - cleaves carboxyl side of Met

Question 6

Edman degradation

Answer 6

sequencing method

phenylisothiocyanate removes first amino acid. Then do another cycle to remove the next AA, etc.

Question 7

Sequencing strategy (steps)

Answer 7

1. determine n-terminus (FDNB thing called Sanger’s reagent)
   - hydrolyzes all peptide bonds, so use another sample for the other steps
2. get rid of disulfide bonds
   - performic acid or dithiothreitol
3. cleave, sequence, establish sequence via overlaps

Question 8

locating disulfide bonds

Answer 8

run two batches (both cleaved or both uncleaved. prob cleave them) batches through a gel

batch 1
-disulfide bonds removed
batch 2
-no removal of disulfide bonds

batch 2 should have two or more bands missing, replaced with larger band on gel

Question 9

understanding (2) sequence pattern representations

Answer 9

logo

• size represents predominance
• stacked on top implies either or

equation
[either or]
{anything but this}

Question 10

right/left handed alpha helices

Answer 10

curl fingers in direction of spiral. Thumb should point up.

Question 11

stability of alpha helix due to…

Answer 11

• maximizing H-bonding
• rigidity (planarity) of peptide bonds
• side chain interactions
• prolines

Question 12

protease

Answer 12

enzyme that catalyzes hydrolytic cleavage of peptide bonds in proteins

ex Trypsin, Chymotrypsin