N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor.
A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, United States of America.
Yaozhong
Y
Zou
William I
WI
Weis
Brian K
BK
Kobilka
D000595:Amino Acid Sequence; D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D003460:Crystallization; D018360:Crystallography, X-Ray; D004082:Dihydroalprenolol / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D014316:Tritium; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D014316:Tritium; D004082:Dihydroalprenolol; D009113:Muramidase