Download Biochemistry I Recitation 3 September 8, 2016

September 8, 2016
Fraction Protonated
Biochemistry I
Recitation 3
1. You are performing a 5 L biochemical
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reaction at a pH of 6.2 using 0.1 M phosphate
0.9
0.8
as a buffer. After several hours, the pH has
0.7
dropped to 5.2. How many moles of NaOH do
0.6
you need to add to restore the pH to 6.2?
0.5
i) Sketch the curve of fraction protonated
0.4
0.3
versus pH for the central ionization of
0.2
phosphate (pKa = 7.2) on the graph on the
0.1
right and mark pH 6.2 and 5.2.
0
0 1 2 3
ii) Also sketch the titration curve for a
monoprotic buffer with a pKa = 7.2 (i.e. the
central ionization of phosphate)
iii) Calculate the fraction protonated at 6.2 and 5.2 using the formula
from class. Do the calculated numbers agree with your sketches?
iv) What is the difference in fraction protonated between pH 5.2 and
6.2 (use your calculated values). How can you use this information
to determine the amount of NaOH required to adjust the pH.
v) Is phosphate the best buffer to use for this reaction?
4
5
6
7
8
9
10 11 12
pH
10
9
8
iii)
7
R= 10pH-pKa
6.2-7.2
-1
R6.2=10
=10
fHA=1/(1+0.1)=1/1.1=0.91
6
5.2-7.2
-2
R5.2=10
=10
fHA=1/(1+0.01)=1/1.01=0.99
These agree with both sketches.
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iv) Difference = 0.99 – 0.91 = 0.08. This is the number of
equivalents of NaOH to add to return the pH to 6.2.
4
Moles NaOH = 0.08eq X 0.1M x 5L = 0.04 moles
0 0.2 0.4 0.6
v) No, the starting pH is just at the edge of the buffer region,
and the reaction moves the pH out of the buffer region. An acid with a lower pKa would better.
0.8
1.0
√Please ask the TA to check your work.
2. Given the following peptide fragments, assemble the complete peptide. You are limited to a seven residue
efficiency during Edman degradation, i.e. any sequence that is 7 long may have come from a longer peptide.
i) State the cleavage specificities in the upper left box.
ii) Use the sequences of the fragments to determine the original sequence.[Hint you need to assemble the
entire sequence using sequence overlaps from each cleavage reaction. The best way to find overlaps is to
identify sites for one cleavage reagent within the sequence of a fragment produced by a different cleavage
reagent.]
Cleavage Specificities:
Chymotrypsin: C1: Met-Pro-Lys-Trp
Cyanogen bromide: Met
C2: Val-Tyr
Chymotrypsin: Phe, Trp, Tyr
C3: Glu-Gly-Gly-Met-Val-Ile-Trp
Trypsin: Lys, Arg
Trypsin:
T1: Met-Pro-Lys
T2: Trp-Glu-Gly-Gly-Met-Val-Ile
Cyanogen bromide: CNBR1: Val-Ile-Trp-Val-Tyr
CNBR2: Pro-Lys-Trp-Glu-Gly-Gly-Met
The correct sequence is: Met-Pro-Lys-Trp-Glu-Gly-Gly-Met-Val-Ile-Trp-Val-Tyr
You can begin with C3, which has a Met, allowing extension in both directions using the chymotrypsin fragments.
C3: Glu-Gly-Gly-Met-Val-Ile-Trp
CNBR1: Val-Ile-Trp-Val-Tyr
CNBR2: Pro-Lys-Trp-Glu-Gly-Gly-Met
T1: Met-Pro-Lys
Can extend to the N-terminus using T1.
√Please ask the TA to check your sequence and explain to them how you obtained your answer.
Before beginning the following problems, please do the following.
A. Go to Blackboard and open the Recitation 3 Links, and place the three pdb files (A,B,C) on your desktop.
B. Click on the link that will bring you to the Jmol page for this recitation (Protein G)
C. Click on the link that will take you to a Ramachandran plot plotting program (RAMPAGE).
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Biochemistry I
Recitation 3
September 8, 2016
4. A structure of Protein G that is displayed in the Jmol window. How many tyrosine and tryptophan residues
does it have? You now have sufficient information to start question 8 on the problem set (See hint 2 on the
Jmol page to obtain the composition of the protein).
There are 3 tyrosine residues and one Trp.
√Please ask the TA to check your work.
5. Ramachandran plots are two dimensional plots that show the conformation of each amino
acid residue in a protein. There are two freely rotatable angle associated with each
residue:
φ (phi) - angle between the N and Cα
ψ (psi) – angle between the Cα and the carbonyl carbon (C).
 Each residue in a protein will give one point on the plot, the location of the point
depends on the phi and psi angles of the residue.
 The contour lines on the Ramachandran plot enclose regions of low (stable) energy.
Your goal is to determine which region of the Ramachandran plot
corresponds to which type of secondary structure.
Navigate to the Rampage Ramachandran Plot server. After you
submit a file you will see a large Ramachandran plot for all of the
residues that you submitted. If you scroll down you will see subplots
for individual residues; General = all but Glycine or Proline, Glycine =
glycine only, Proline = proline only. The colored regions of these
plots indicate low energy regions = favorable van der Waals. White
areas indicate high energy regions = unfavorable van der Waals. You
only need to focus on the first (larger) plot.
a) Submit the A.pdb file to the server. Mark on the above
Ramachandran plot the location of the points for this structure.
Also view A.pdb in Jmol – what is its secondary structure? Label
your Ramachandran plot accordingly. This is the structure of a


β
αL
αR
right-handed alpha-helix.
b) Submit the B.pdb file to the server. Mark on the Ramachandran
plot the location of the points for this structure. Also view B.pdb in Jmol - what is its secondary
structure? Label your Ramachandran plot accordingly. B.pdb are two beta-strands.
c) How is the structure of C.pdb different from B.pdb? Submit the C.pdb file to the server and plot the new
points that are unique to C.pdb. What does this tell you about the phi & psi angles for the residues that
were added to B.pdb to give C.pdb. The additional residues in C.pdb are in the tight turn that
joins the two b-strands. The residues in the turn have non-regular phi-psi angles, but still
in the low energy regions of the plot.
√Please ask the TA to check your work.
6. Load the helix structure in Jmol and:
a) Determine the length of the helix both in terms of residues and distance. When measuring the distance,
use the same atom on the two residues (e.g. N) (you probably want to remove the sidechains). 15
residues long, 21 A long, so about 1.4 A/residue.
b) Identify a mainchain hydrogen bond in the helix, what are the residue numbers for the N and the C=O
(Note: there are no hydrogens on this structure, infer the position of the NH hydrogen). Select the N
above a C=O group – note that the hydrogens are not shown.
c) How many residues/turn in the helix? 3.6
d) What direction do the sidechain point? Outwards and slightly downwards.
√Please ask the TA to check your work.
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