Methods for Stabilisation and Crystallisation of Unstable Eukaryotic Membrane Proteins. (360G-Wellcome-087731_Z_08_Z)

Determination of membrane protein structure requires crystallisation of purified protein. The main problem in obtaining crystals of many important eukaryotic or human membrane proteins such as G-protein-coupled receptors (GPCRs) or ion channels is their poor stability after detergent solubilisation, which is essential for purification and crystallisation. Recent work by the Tate group at LMB, following earlier work by Bowie, showed it is possible to increase greatly the stability of membrane pro teins by limited mutagenesis while retaining key functions, termed conformational thermostabilisation. We propose to develop further the technology involved by tackling three particularly challenging eukaryotic membrane protein structures, each posing new problems that require extensions of the existing very promising results. The structures are (a) the agonist-bound beta1-adrenergic receptor, an unstable conformation that binds ligands weakly (b) the antagonist state of human muscarinic M1 acet ylcholine receptor in which the third cytoplasmic loop is very large, and (c) the human voltage-dependent sodium channel, NaV1.7, which is a very large molecule with multiple functional conformations. Development of more rapid assays for binding of weaker ligands to lower levels of expressed protein will allow the method of conformational stabilisation to be extended so that it can be used on many more membrane proteins..