ࡱ> 685 ?bjbj 4$__?  4MpaaaaaPPPceeeeee$T  #jPPPPPaaP^aacPc=X O0t#(t#t#PPPPPPP(PPPPPPPt#PPPPPPPPP : MSc, MBio or MIBTP mini research project - Napier Title of research project: Binding site mutants: matching amino acid side groups to ligand selectivity Academic supervisor: Prof Richard Napier Other supervisors (e.g. postdocs): Dr Martin Kubes, Dr Charo del Genio 1. Background and project aims (max 500 words) Hormones are mobile molecules that carry signals to coordinate growth and initiate responses to environmental cues. A hormone receptor protein acts as a molecular switch, initiating a chain of events that ultimately leads to developmental responses. We study a protein called TIR1 which we express in an insect tissue culture system for purification by IMAC and other affinity chromatographies. TIR1 binds the hormone and in doing so the hormone completes a binding site for a second protein, a transcription factor. This binding event is unusual in that the hormone is acting as 'molecular glue'. There is no conformational change, the hormone sits at the interface of TIR1 and a separate peptide known as the degron domain on the transcription factor. The structure of TIR1 is published and we use TIR1 as the template for structure-led drug design and development. Intriguingly, the binding site is at the base of a deep binding pocket and we believe the walls of the cavity on the approach to the binding site help select the correct, active ligands, and filter out similar, but inactive competitors. This type of gatekeeper selection is novel. In this project you will examine how specific amino acids at the sides of this deep binding pocket contribute to this gatekeeper activity. You will clone a small set of mutations into TIR1 and then generate recombinant baculovirus lines for each mutant. Using these to drive protein expression in Sf9 cells in culture, you will purify mutant versions of TIR1 and screen sets of compounds for affinity and selectivity. You will learn SPRT as a convenient, yet advanced method for evaluating binding of each version of the protein. You have the chance to be part of the team describing such molecular gatekeepers for the first time. 2. References Tan et al., (2007) Nature 446, 640-645. Calderon Villalobos LIA et al., (2012) NATURE CHEMICAL BIOLOGY, 8, 477-485. Lee S, et al., (2014) ACS Chemical Biology, 9 (3), pp 673682. Uzunova et al., (2016) 3. Discipline-specific skills and transferable skills the student will develop during the project Tissue culture, protein expression using eukaryotic cells (baculovirus system), protein purification using affinity tags including IMAC, biochemical and biophysical techniques such as SPR and ITC, pharmacological techniques such as dose-response, structure-activity profiling, molecule docking etc. Transferable skills: teamwork, communication skills, organisation and time management. - how often will you meet the student? Daily.  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