Tutorial
Before simulating fluorescence decays and fluorescence quenching you have to ensure that the structure of interest is in an appropriate data format. Additionally, crystal water potentially contained in the structure as provided by the protein databank (PDB) should be removed. This is exemplified by a structure (PDB-ID: 172L) of T4 lysozyme (T4L). As can be seen in Fig. 2 the water contained in a crystal structure may exclude a significant fraction of the surface otherwise accessible by the dye. The crystal water should be removed from the structure as potentially accessible quenching sites are otherwise inaccessible.
Structure of T4-lysozyme (shown in green) and the crystal water contained in the structure contained in the protein databank (PDB)
Next, the protein structure should be protonated and the naming of the atoms has to be adapted to following the naming convention of the “PARSE” forcefield. This can be accomplished by uploading the protein structure to the PDB2PQR-webserver.
Download the resulting PQR file and save it as PDB-file. This PDB-file can be opened in QUEST by clicking on (…) highlighted in the figure below by the red number “1”. Next, adjust the parameters to your needs and click on the “update” button to calculate a BD-trajectory and the corresponding fluorescence decay. These steps are shown in the figure below.
