Solutions to Selected End of Chapter 3-Part 3 Problems
18. Brian peptide Leucine Enkephalin Sequence Determination.
Data: a. 6M HCl hydrolysis at 110oC showed G, L, F, Y in a 2:1:1:1 ratio.
This means the amino acid composition had to be 2G, L, F, Y; or 4G, 2L, 2F, 2Y, or….
b.Treatment with FDNB followed by complete HCl hydrolysis showed the 2,4-DNP derivative
of Y and no free Y could be found.
This means that Y was N-terminal. And, with no other Y’s, the peptide could only be 2G, L, F,
Y. So, at this point the peptide is: Y _ _ _ _ .
c. Chymotrypsin digestion of the peptide showed free Y and L, plus a tripeptide having F and
G in a 1:2 ratio.
This means several things. First, because chymotrypsin cuts only on the C-terminal side of
aromatic amino acids, it is logical to get free Y from the N-terminal amino acid. And, second L
has to be C-terminal with F being right next to it based again on chymotrypsin’s specificity: So
at this point the peptide is Y _ _ F-L. And because there are 2 G’s, the final peptide
sequence is Y-G-G-F-L.
19. This is one of the classic sequencing problems. It has an unusual, non-protein amino acid,
ornithine which is part of the Urea Cycle (we will see in Chapter 18). Ornithine is easy to know, it is
one methyl shorter than Lys, but otherwise like it having a amino-R group, we can give it the one
letter amino acid code “O”.
Data: a. Complete acid hydrolysis followed by amino acid analysis showed L, O, F, P. V in equimolar
amounts.
b. The molecular weight was about 1,200.
This means that there are two of each amino acid because the minimum MW (one of each)
would be 550 which if doubled would be close to 1,200. And, definitely not 1 or 3.
c. The peptide was resistant to carboxypeptidase, an exoenzyme hydrolyzing off one amino
acid at a time from the C-terminal end.
That means this peptide seems to be lacking a C-terminal amino acid.
d. Treatment of the peptide with FDNB yielded the 2,4 DNP derivative of O, with the 2,4 DNP
attached only to the R-amino group, not the α-amino group. This means there is no N-terminal
amino acid.
And, taking “c” and “d” together means the peptide is likely circular. Circles do not have ends.
e. Partial hydrolysis of the peptide (meaning cutting the peptide at random points) along with
sequencing each smaller peptide product produced:
L-F
F-P
O-L V-O
V-O-L
F-P-V
P-V-O
Putting these together, overlapping sequences:
L-F
F-P
F-P-V
The minimum sequence is then: L-F-P-V-O, and then the
P-V-O
complete sequence making a circle is then:
V- O
O – (L)
21. Sequence comparisons: the molecular chaparone, Hsp90 contains a signature sequence
of 10 amino acids which allows for identification of similar proteins from protein sequence
databases. One of the signature sqeunces is:
Questions:
a. Which amino acids are invariant (highly conserved) among species of Hsp90?
Answer: Y (1), F (7) , R (9) and E (10).
b. Which positions have positively charged amino acids, and which predominates?
Answer: Positions 4 and 9: K is more common but not invariant, R is invariant.
c. Which positions are negatively charged, which amino acids dominate?
Answer: Positions 5 and 10, both have glutmate.
d. Which position can be any amino acid eventhough one dominates?
Answer: Position 2 serine dominates, and all the other amino acids are beneath
it, though that might be hard to see, but the all group is taller and part is wider.
22. Chromatographing three polypeptides. The three polypeptides are:
1. ATKNRASCLVPHGALMFWRHKQLVSDPILQKRQHILVCRNAAG.
2. GPFYGDEPLDVHDEPEEG.
3. PHLLSAWKGMEGVGKSQSFAALIVILA.
Questions and Answers:
a. Which one would move the slowest through an ion exchange column having beads with a
positive charge?
Answer: Polypeptide 2. It is loaded with negative charged R groups.
b. Which one would move the slowest through an ion exchange column having beads with a
negative charge?
Answer: Polypeptide 1 has 6 positively charged groups (Ks and Rs).
c. Which one would move the slowest through a gel filtration column?
Answer: Polypeptide 2, it is the smallest so it would go through more of the bead volumes.
d. Which one has an ATP binging motif show by:
Answer: Polypeptide 3 has the sequence G-x-x-G-x-G-K-S