Prof Maria Sunnerhagen
Molekylär bioteknik, IFM, LiU
BIOMEASUREMENT TECHNOLOGIES 2011- Class 2.
Exercise 1.
The protein Sm14 is a fatty acid binding protein and a promising vaccine candidate against
the parasites Fasciola hepatica and Schistosoma mansoni. DAUDA, a fluorescent fatty acid
analogue (11-(dansylamino)-undecanoic acid) is employed to study the ligand binding
properties of the Sm14 protein (Ramos et al., Journal of Biological Chemistry 2003).
a. Explain why the fluorescence intensities are different for the different cases in the
experiment below (use a scientifically specific language).
b. Explain how the results from the figure can be used to design an experiment where the
affinity of linoleic acid can be determined!
Prof Maria Sunnerhagen
Molekylär bioteknik, IFM, LiU
Exercise 2.
TBP binding to the TATA-box is central to protein translation in eukaryotes, since this
recognition process induces binding of the RNA-polymerase complex. In this experiment,
fluorescence labelling has been empolyed to study the TBP-TATA complex (Liu & Schepartz,
Biochemistry 2001).
TBPc has no native tryptophane (W). Therefore, a mutation was made where a W was
introduced in one half of the protein (Figure A). Using organic synthesis a DNA duplex was
designed where one of the bases on a specific position in the TATA-box sequence was
exchanged for a synthesized DNA-base analogue with a fluorescent tag (Figure B). Molecules
indicated with ball-and-stick in figures A and B represent the two fluorophores.
a. Which type of fluorescence experiment can be performed with these protein- and
DNA constructs? Why does this type of experiment require fluorophores on both the
protein and the targetted DNA?
b. What type of information would be obtainable using a fluorophore on the TBP protein
alone, or the TATA-DNA alone?
c. Describe and explain the results in figure C, where the TATA_box was titrated with
TBP-E188W.
Black line: E188W alone. Grey line: E188 with unlabelled DNA. Dashed black line: E188
with TATA22down. Dashed grey line: E188W with TATA22up.
Prof Maria Sunnerhagen
Molekylär bioteknik, IFM, LiU
C
B
Prof Maria Sunnerhagen
Molekylär bioteknik, IFM, LiU
Exercise 3
This study concerns a folding variant of the human protein α-lactalbumin, which induces apoptosis in
cancer cells. The folding variant is called HAMLET. The study was performed to investigate whether
there are any structural grounds as to why human apo-α-lactalbumin (HAMLET) can reduce tumor
size, whereas recombinant enzyme lacks this property. Solid lines show native α-lactalbumin and
dashed lines show the HAMLET-proteinet.
a) Which biomeasurement methods have been used in Figure A and B respectively?
b) Why have different wavelengths been used in A and B? What is measured in these
wavelength regions?
c) What conclusions can you make on protein conformation based on figures A and B?
d) What type of CD measurement would you want to add to this study for completeness?