Université Joseph Fourier - Grenoble 1
Year 2012-2013
Bio 121 Final Exam (Session 1)
Duration: 2h
No document, no calculator, no cellular phone.
Answers have to be written directly on the exam subject
Total points: 100
PART C (25 points)
(Time recommended - 30 min)
A researcher wants to characterize the interaction between a protein A and a peptide
B.
The analysis of the purified protein A by acrylamide gel electrophoresis run in native
conditions, shows a single band with an apparent molecular weight of 45 kDa. When
the protein A is analyzed in the presence of SDS it gives this SDS-PAGE below:
1- What is the major difference between the two methods, (native versus SDS)?
Justify your answer. (1.5 pt)
1
2- Describe the results of the SDS-PAGE gel and draw a schematic representation of
the native structure of protein A after having estimated the size of the proteins on the
electrophoresis profile. (5 pts)
3- If protein A (which is pure) had been analyzed by gel exclusion
chromatography, how many elution peaks would you have expected on the
chromatogram and what would be the estimated apparent molecular mass(es) ? (1.5
pt)
The researcher then aims at isolating the region of protein A that interacts with
peptide B. He thus attaches the B peptide (marked by a fluorescent probe on its Nterminal extremity) on the A protein using a reticulating reagent. This reticulating
agent transforms any electrostatic interaction implicated in the association between
two proteins, into a covalent bond. After this treatment, protein A is incubated with βmercaptoethanol, suspended in a buffer at pH 7 and loaded onto an anion
exchange column. A pH gradient from pH 3 to pH 7 is then applied onto the column.
The obtained elution profile is presented below.
2
wash
Begining of the
pH gradient
wash
Begining of the
pH gradient
4- Describe the principle of the gel exclusion chromatography (1.5 pt)
5- Each of the 3 peaks contains a polypeptide chain X,Y or Z. Indicate the name of
each peptide in the frames placed above the chromatogram peaks , knowing that pHi
X = 4, pHi Y = 6 and pHi Z = 8. Comment on the basis of the given data and use a
scheme for your explanation. (5.5 pts).
3
This protocol allowed the isolation of the polypeptide chain of protein A that is
attached to peptide B. The tri-dimensional structure of protein A attached to peptide B
is represented below:
6- Which polypeptide chain of protein A does interact with peptide B? What are your
arguments to justify this conclusion? (1 pt)
7- What type of interaction (indicated by the arrows on the structural model above)
does take place between the residues Asp7 and Glu 11 of peptide B and protein A,
knowing that this interaction is dependent on the pH? Comment your answer (2 pts)
4
8- What kind of amino acid residues of protein A can interact with both residues Asp7
and Glu11? (1.5 pts)
9- Give the semi-developed formula of residues Asp7 and Glu11 at pH7. Would this
interaction between peptide B and protein A be maintained at pH1.5? Comment your
answer.
(3.5 pts)
Asp7
Glu11
10- What type of secondary structures do characterize peptide B and protein A? (2
pts)
5