I have so much to say about everyone in the group, but I'll keep it short. He has been both motivating and supportive during our last months here and our departure will significantly lower the average age of the North Bay. Matt Rienzo and Mike Post have been two of my best friends in the group.
In Chapter 2, the role of a conserved aromatic microdomain, predicted in the D3 dopamine receptor, is investigated in the closely related D2 and D4 dopamine receptors. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are being investigated to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the previously observed interactions are conserved in the GluCl receptors, but caution should be exercised when extrapolating from structural data.
Chimeric synthesis and mutagenesis reveal that the C-terminal portion of the M4 helix and C-terminus contribute to the formation of the uncoupled state, where ligand binding is unable to trigger channel gates. We conclude that the M4 helix structure and/or membrane dynamics are likely the cause of ligandone sensitivity in this receptor and that the M4 helix plays an important role in the activation process.
Introduction
G protein activation results in the exchange of GDP for GTP and the separation of Gα and Gβγ subunits. However, in the presence of suppressor tRNA (a tRNA with an anticodon designed to recognize the TAG stop codon) a full-length protein containing the appropriate unnatural amino acid at the site of interest is produced. In the case of ligand-gated ion channels, the observed current is directly related to the channel activity.
Torrice, M.M., Bower, K.S., Lester, H.A. and Dougherty, D.A., Probing the role of the cation-pi interaction in the binding sites of GPCRs using unnatural amino acids. In the D3R crystal structure, the side chain of C3.36 points directly to the face of the aromatic ring of W6.48. To further examine the nature of the interactions identified through mutant cycle analysis, unnatural amino acid mutagenesis was performed.
A linear trend indicates the existence of an electrostatic interaction with the surface of the residue examined. However, W6.48 is the only aromatic microdomain residue considered here that contributes to the aromatic cage. Lummis, S.C.R., Beene, D.L., Harrison, N.J., Lester, H.A. and Dougherty, D.A., A cation-pi binding interaction with a tyrosine in the GABA(C) receptor binding site.
This is likely to complicate conventional and unnatural amino acid mutagenesis experiments in the future due to the need to optimize subunit biases for each mutant.
Wild type eGFP expression B) eGFP expression from HSAS suppression at A37TAG using the Neon transfection system. Confocal image is at both the laser power used to
Limapichat, W., Lester, H.A., and Dougherty, D.A., Chemical scale studies of the conserved Phe-Pro motif in the Cys loop of Cys Loop receptors. Functional assessment of key interactions evident in the structure of the eukaryotic Cys-loop GluCl receptor. In the present work we decided to determine if the main structural features of C.
Residues in the ion-conducting pore are highlighted in blue and the location of 0' arginine and 20'. Side chains of the GluClα crystal structure were mutated to the corresponding GluClβ side chains in PyMol. Hydrogen bond network between (A) C loop of primary subunit and F loop of complementary subunit B loop of primary subunit and E loop of complementary subunit (B) B loop of primary subunit and E loop of the complementary subunit.
In summary, a series of interactions in the GluClβ receptor predicted by the crystal structure of the homologous GluClα receptor were investigated. Lummis, S.C., D, L.B., Harrison, N.J., Lester, H.A., and Dougherty, D.A., A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor. Despite the ability of the GluClα receptor to bind glutamate, the receptor is not activated by glutamate in the absence of allosteric modulators such as ivermectin.
Mutagenesis of the GluClβ receptor, including amino acid deletions, was performed using the QuikChange protocol (Stratagene). The properties of the C-terminus and the M4 helix are responsible for uncoupling of ligand binding and channel gating to GluClα. The numbering for mutations made in the M4 helix starts at the N terminus of the M4 helix as residue 1 (threonine) and continues to the C terminus of the GluClα receptor and ends at residue 40 (phenylalanine) (Figure 5.4).
Two possible hypotheses to explain this phenomenon are either the properties of the M4 helix in the X. The GluClα receptor contains a Phe-Pro motif near the center of the M4 helix which is absent in the GluClβ receptor. Properties of the receptor, including the M4 helix, dictate the length requirements of the C-terminus.
Conversely, truncation of the terminal 9 amino acids of the GluClα/β-M4 chimeras had no effect on receptor function. This is supported by a large loss of functional response to glutamate in the NL mutation of the -9 residue (from the end of the C-terminus) in the GluClβ -7 C-terminal truncation (Table 5.3). Green and purple residues are conserved between the GluClα and β receptors, purple residues are located in the 17–34 region of the M4 helix.
The crystal structure of the GluClα receptor shows a kink and bulge approximately in the middle of the M4 helix.
