Task 4 15 December 2020 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. Tthis 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 the videotutorial here. ][10 QK F F SV+= X1 X2 rezervoár Koncentrace akrylamidu [M] 0 0.1 0.2 0.3 0.4 0.5 1 Dikunová Alžbeta A 944 911 891 870 853 834 B 944 794 697 621 560 510 2 Dzurov Matej A 977 943 922 901 883 864 B 977 822 722 643 580 528 3 Faturová Jana A 940 908 887 867 850 831 B 940 791 694 619 558 508 4 Gašparik Norbert A 951 918 898 877 860 841 B 951 800 703 626 565 514 5 Hesko Ondrej A 960 927 907 886 868 849 B 960 808 709 632 570 519 6 Jahodová Kateřina A 986 952 931 910 891 872 B 986 830 729 649 585 533 7 Kameniarová Michaela A 938 906 886 865 848 829 B 938 790 693 617 557 507 8 Konečná Kateřina A 957 924 903 882 865 846 B 957 805 707 630 568 517 9 Korytářová Anna A 986 952 931 910 891 872 B 986 830 729 649 585 533 10 Kozeleková Aneta A 975 942 921 899 881 862 B 975 821 720 642 579 527 11 Kubinyiová Lenka A 981 947 926 905 886 867 B 981 825 724 646 582 530 12 Kůřilová Eliška A 957 924 903 882 865 846 B 957 805 707 630 568 517 13 Lysáková Klára A 961 928 908 887 869 850 B 961 809 710 633 571 520 14 Mikšátková Barbora A 855 834 813 794 776 758 B 855 732 639 568 510 464 15 Nováková Barbora A 943 911 891 870 853 834 B 943 794 697 621 560 510 16 Prabhullachandran Unnikannan A 978 943 922 901 883 864 B 978 822 722 643 580 528 17 Procházková Markéta A 941 908 887 867 850 831 B 941 791 694 619 558 508 18 Šimek Jan A 953 918 898 877 860 841 B 953 800 703 626 565 514 19 Tužinčin Dávid A 962 927 907 886 868 849 B 962 808 709 632 570 519