Protein Structure and Function Lecture 5 Notes: Protein Engineering Part 2 - Natural binding proteins can be adapted to be used as a biosensor by introducing reporter groups. For example, bacterial periplasmic binding proteins are naturally occurring maltose biosensors. Furthermore, metalloproteins can be used for the selective detection of trace metals in basic biological research and environmental/clinical chemistry. Alternatively, we can rely on GFP-based molecular sensors or antibody-controlled ion channels. - Two strategies have been used to design modular protein-engineering systems for biosensor development. Firstly, find a protein with the appropriate specificity (e.g. metal-binding) and introduce a signal-transduction function. Most commonly, this is covalently coupled with a reporter group (e.g. a fluorophore). Alternatively, identify a protein with a particularly efficient intrinsic signal-transduction function and construct appropriate binding sites. - Maltose-binding protein and glucose-binding protein can be modified by introducing two fluorescent groups at two key positions. When a ligand is bound to the protein, there is a conformational change, bringing the two fluorescent groups together, causing fluorescence resonance transfer (FRET). This can be detected under a microscope so the concentration of maltose at the location of the protein can be detected. - The zinc-finger motif is a metal-sensitive protein which, when bound to zinc, has a dramatic conformational change (becomes very tightly bound). A fluorescent motif can be inserted to change the fluorescent properties of the molecule. - Two GFP domains (one modified to be a cyan-emitting protein) will have a FRET interaction if brought together. If the domains are connected by a calmodulin-binding peptide, calmodulin can bind to calcium, changing the conformational shape of the recombinant polypeptide, bringing the GFP domains together. This enables intracellular measurement of calcium- concentration. - Enzymes bind substrate and act as catalysts to convert them to product, usually via a transition state. Catalytic antibodies (abzymes) can be generated to have complementary binding sites to substrate transitional states. Therefore, when the engineered antibody (i.e. new enzyme) binds, it induces the transitional state in the substrate (i.e. catalysis occurs). - Abzymes have potential applications as artificial enzymes/'designer' catalysts. For example, abzymes which cleave the viral coat proteins of HIV have been isolated. - Cancer cells may contain unique determinants on their surface (e.g. tumour cell antigens) which may be lacking in normal cells. Using a traditional approach, antibodies that specifically bind to tumour cell antigens could be used to deliver drugs directly to a tumour. However, abzymes (recombinant antibodies with two distinct antigen binding sites) could be designed to bind one site to a tumour antigen with high affinity, whilst the second site catalyses the cleavage of a prodrug (non-toxic precursor of a cytotoxic drug). - Protein engineering relies on known information (e.g. structure) about an existing protein to change (mutate) certain genes to alter its function. Examples of this are improved enzymes, Ab engineering and transcription factors. However, this process relies on using a model which is very close to the desired product. - On the other hand, protein design is a rational design technique