Biochemistry Practical - Experimental Layout.
2-aminoquinoline (2AQ) can bind to a Tec SH3 domain in humans. It does this by the formation of a salt bridge using a resonance delocalised negative charge on aspartic acid present on an RT loop in close proximity to tryptophan 215 and a resonance delocalised positive charge on 2AQ. Binds with greater affinity at lower pH, supporting salt bridge assumption. Needs to form a good pi-pi stack between Trp and 2AQ. Both aromatic rings have delocalised pi electron clouds, one (Trp) which is slightly negative and the other (2AQ) which is slightly positive. Hence polarity exists. .
The protein sequence bound by SH3 domains is proline rich, often of sequence PxxP. Three binding pockets on flat hydrophobic ligand-binding surface, two occupied by hydrophobic-proline dipeptides. There are 511 SH3 domains across human proteins. Some have 5-6 such domains. They serve as scaffolds, and have an active role in signal transduction, but have no biological function (such as phosphorilation). Some proteins have both SH2 and SH3 domains, conferring an adaptor function. Can block signal transduction events using drugs, ie treat diseases. .
Need to get a low kd of drug, so that there is strong association ensuring deactivation. kd represents the ligand concentration at which 50% of SH3 protein has bound. Need to interact target SH3 domain protein with a natural ligand, then progressively increase synthetic ligand concentration. Analysis of detachment kinetics yields the strength of synthetic ligand (2AQ) binding, and also simulates 'in vivo' results 'in vitro'. Need to flourescently label the natural ligand, as we are monitoring its dissociation from the SH3 domain with increasing competitor (2AQ) binding (non-competitive or competitive binding?). See end of document for graphs detailing binding of ligands to the SH3 domain.
Experiment has two steps:.
1) Synthesis of protein of interest by:.