| Popis: |
The fluorescence emission of the single tryptophan (W233) of the mutant protein DD carboxypeptidase from streptomyces is characterized by a red edge excitation shift (REES). This phenomenon is an indication for strongly reduced dynamics in the environment of the tryptophan residue, which has a very low accessibility to the solvent. The Stokes shift however, shows an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states which can be identified using the Dead End Elimination (DEE) method. The three individual lifetimes increase with increasing emission wavelength. This is interpreted as each individual lifetime being an average lifetime on its own, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time resolved anisotropy measurements, which demonstrate dynamics within the rotamers but not among the rotamers and by maximum entropy analysis producing distributions that shift with the emission wavelengths. The maximum entropy distributions can also be fitted using a gamma-function analysis, again indicating a dynamic component next to a static heterogeneity. Advanced DEE calculations together with MD simulations indicate the existence of two minima (i.e. substates) within one particular rotamer, with frequent transitions. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states, one of which is further subdivided into two substates, with different emission spectra. |
