title: A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs.
abstract: Determination of high-resolution, three-dimensional structures of transmembrane proteins (TMPs) has, in many cases, only been accomplished through the use of stabilized variant forms of the proteins being studied. For the important G protein-coupled receptor superfamily, this has most often been achieved by inserting a stable soluble protein, such as T4 lysozyme (T4L), in an internal loop of a receptor. However, creation of such fusion proteins generally results in loss of the ability of receptors to activate their cognate cytoplasmic G proteins. Furthermore, the criteria for designing fusions that minimally perturb receptor structure are not well established. We describe here a method for creating a library of receptor variants containing T4L inserted into an internal loop at varying positions and as replacements for varying amounts of the original receptor sequence. We also describe methods for screening for variants displaying maximal expression levels, ligand binding capacity, and signaling function. When applied to the yeast α-factor receptor, Ste2p, this approach allowed recovery of well-expressed receptor variants containing internally fused T4L that retained nearly normal signaling function. The approach we describe can be readily adapted to creation of stabilized fusions of other TMPs expressed in yeast or other expression systems.
authors.affiliation: Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mathew
authors.affiliation: Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. Electronic address: mark_dumont@urmc.rochester.edu.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dumont
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D009113:Muramidase / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics*; D051964:Receptors, Mating Factor / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D012995:Solubility; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D009113:Muramidase
keywords: G protein-coupled receptors; Membrane proteins; Pheromone receptor; Protein stability; Random screening; X-ray crystallography; Yeast genetics