| Here I present work on two unique proteins united by a general design principle: the introduction of novel functions into well known structures. In one project we endeavored to design a fluorescent protein-based biosensor specific for inorganic mercury (II). The introduction of a cysteine residue at position 205, located in close proximity to the chromophore, converted the green fluorescent protein (GFP) into a highly specific biosensor for this metal ion. The resultant fluorescent properties, with respect to the metal concentration, permitted real-time detection into the low nanomolar ranges. In a first application it was shown that this sensor can be used to observe mercury uptake from the environment by prokaryotic and eukaryotic organisms alike. This rapid uptake was even detected if the sensor-expressing cells were shielded by layers of surrounding tissues, suggesting that neither individual cell walls nor tissue layers provided a substantial barrier for the metal. Furthermore, the suitability of this biosensor for the direct imaging of mercury uptake through the food chain was demonstrated. To our knowledge, this engineered protein was a first example of a biosensor that allowed for the non-invasive and real-time imaging of mercury uptake in a living cell.;In a second project, a method for engineering specific allosteric functions into a protein was developed and termed "Combinatorial Annealing". The fundamental experimental principle is based on a deliberately destabilized protein to engineer effector controlled conformational changes. The design process is analogous to an annealing procedure where the "melting" step is the designed loss of protein stability, and through random mutations the stability can be restored in a strictly function specific manner, thus resulting in an "annealed" structure. By the use of substrate-affinity chromatography, responsive mutants of glutathione-S-transferase were rapidly identified. The general applicability of the method was shown by generating (1) pH-dependent substrate release, (2) indole-specific substrate release, and (3) "competitive" activation by imidazole. |
