Task 4 10 May 2017
Mutant identification
Your first real task in biotechnological laboratories BTP (Biotechnology for purification) is
to distinguish between two mutant variants X1 and X2 of a protein which is used for the
decomposition of toxic substances in drinking water. Accidentally, labels of reaction
reservoirs with enzymes were confused. You have found out that both types of enzyme
contain one tryptophan. Additionally, you know that tryptophan in case of mutant X1 is
placed much closer to the surface. Therefore, X1 is in contact with surrounding solution
more than X2.
The addressing of this task is important for the supply of drinking water to residents in an
area affected by drought. You can use your knowledge of fluorescence quenching for the
problem solution. You remember that the protein with a tryptophan located on the surface
can be determined from the dependence of fluorescence intensity on the concentration of the
quencher. For quenching fluorophore, the basic Stern-Volmer equation can be applied:
where F0 is fluorescence intensity in the absence of quencher, F is the fluorescence intensity
in the presence of quencher, KSV is Stern-Volmer constant and [Q] is the concentration of the
quencher.
You have carried out measurements of fluorescence intensity of proteins taken from
reservoirs A and B. The fluorescence intensity was measured in the absence of the quencher.
Then you measured fluorescence decrease after gradual addition of quencher (acrylamide).
The obtained values of fluorescence intensity are in the table below.
Plot the dependence of relative fluorescence intensity decrease on the acrylamide
concentration in the form of Stern-Volmer graph and answer the following questions:
1. Is acrylamide a dynamic or static quencher?
2. What are constant KSV values corresponding to each mutant of the enzyme?
3. Determine in which reservoir X1 mutant is located.
Please send me your answers together with Stern-Volmer plot for A and B via email.
Correct answer = 0.5 point
Note: Determine Ksv as a slope of linear regression according to a videotutorial here.
https://is.muni.cz/el/1431/jaro2017/S2006/um/Tasks/Regression_Linear_Excel.wmv
][10
QK
F
F
SV+=
X1 X2
reservoir
Acrylamide concentration [M]
0 0.1 0.2 0.3 0.4 0.5
1 Akhmetgalieva,
Valentina
A 944 911 891 870 853 834
B 944 794 697 621 560 510
2 Alispahic, Elma A 977 943 922 901 883 864
B 977 822 722 643 580 528
3 Atatri, Sura S. M. A 940 908 887 867 850 831
B 940 791 694 619 558 508
4 Janovič, Tomáš A 951 918 898 877 860 841
B 951 800 703 626 565 514
5 Lobello, Cosimo A 960 927 907 886 868 849
B 960 808 709 632 570 519