%0 Generic
%A Vaiana, Andrea C.
%D 2004
%F heidok:4454
%K molecular modeling , molecular dynamics , fluorescence quenching , force field parameters , cancer
%R 10.11588/heidok.00004454
%T Towards the Understanding of Fluorescence Quenching Mechanisms : Molecular Dynamics Simulations of Dye-Quencher Interactions in Biomolecular Systems
%U https://archiv.ub.uni-heidelberg.de/volltextserver/4454/
%X The presence of antibodies directed against p53 in human blood serum is a specific and independent marker of cancer. The present thesis is a simulative study of two fluorescent dyes (rhodamine 6G and MR121) used in the development of fluorescence-based immunoassays for the detection of p53 antibodies. The "selective" fluorescence quenching property of tryptophan residues present in dye-conjugated peptide chains enables monitoring of conformational dynamics and antibody binding events by means of fluorescence spectroscopy. The molecular mechanisms of the quenching of fluorescent dyes are mostly unknown. Here MD simulations are used in combination with existing results from ensemble fluorescence experiments in order to obtain predictive theoretical insight into dye/quencher interactions. A new automated refinement method was developed for deriving reliable molecular mechanics force field parameters for small- to medium-sized molecules using reference data from high level ab initio quantum chemical calculations. Using this method CHARMM force field parameters for the two dyes were derived. The parameters were then used to perform MD simulations on two simplified, but realistic dye quencher systems: MR121/TRP and R6G/TRP. Results of these simulations have given important insight on the bimolecular interactions between the dyes and the quencher. A quantitative interpretation of the quenching mechanism based on it’s dependence on dye/quencher distance and orientation has emerged. These results were then applied to the interpretation of simulation data of an epitope from the tumor suppressor protein p53 which was labelled at the N-terminus first with MR121 and then with R6G. In all phases of the thesis care was taken to confront and/or combine the theoretical results with available experimental data.