title: Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs.
abstract: The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactory-related receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (M(W) = 178.23) and myrtenal (M(W) = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies.
authors.affiliation: Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.
authors.forename: Karolina
authors.initials: K
authors.lastname: Corin
authors.forename: Horst
authors.initials: H
authors.lastname: Pick
authors.forename: Philipp
authors.initials: P
authors.lastname: Baaske
authors.forename: Brian L
authors.initials: BL
authors.lastname: Cook
authors.forename: Stefan
authors.initials: S
authors.lastname: Duhr
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Wienken
authors.forename: Dieter
authors.initials: D
authors.lastname: Braun
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zhang
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics*; D000081008:Bicyclic Monoterpenes; D002630:Chemotactic Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013729:Terpenes / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000081008:Bicyclic Monoterpenes; D002630:Chemotactic Factors; D003902:Detergents; D008024:Ligands; C473192:OR1D2 protein, human; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; D013729:Terpenes; C414724:VN1R1 protein, human; D014764:Viral Proteins; C061545:myrtenal; D009113:Muramidase