I appreciate his friendship and look forward to seeing what he does in the future. Hydrogen bonding involving the agonist appears to be important for ligand binding in the muscle-type receptor, but not in the α4β4 receptor.
Ligand-gated ion channels
The five subunits are arranged pseudo-symmetrically around a pore lined by the second transmembrane helix, and ligand-binding sites are located at subunit interfaces in the extracellular domain. Our knowledge of nAChR ligand binding sites has been enhanced by the structures of invertebrate acetylcholine binding proteins (AChBPs) (Figure 1.1B).
G protein-coupled receptors
The major ligand-binding pocket is typically located between TMs 3, 5, and 6 on the extracellular half of the receptor, beneath extracellular loop 2 (EL2). Although the specifics of ligand binding vary from receptor to receptor, increasing evidence from both crystallography and biochemical studies suggests that receptor activation among the different classes of GPCRs involves common conformational changes in the transmembrane helix bundle, causing activation of the G protein at the receptors becomes possible. The intracellular surface of the receptor.18,19 Briefly, agonist binding induces subtle changes at the extracellular half of the helical bundle, accompanied by a more pronounced spreading of the intracellular end, particularly of TM6 and TM7.
Methods for interrogating ion channel and GPCR function
Electrophysiology as an assay for ion channels and GPCRs
For both GPCRs and ligand-gated ion channels, receptor function is analyzed by constructing a dose-response curve from the instantaneous responses to progressively increasing agonist concentrations (Figure 1.5A,B). EC50 is a metric of receptor function; mutations that increase the EC50 relative to the wild-type receptor (Figure 1.5B, red curve) are called "loss-of-function" since it is a larger agonist. concentration required for the same response, while those that lower the EC50.
Unnatural amino acid mutagenesis
To specifically incorporate unnatural amino acids, we use the nonsense suppression method for ribosomal incorporation.21 The mRNA code corresponding to the amino acid of interest is replaced with a nonsense (stop) codon and a suppressor tRNA with the corresponding anticodon is given. For unnatural amino acid mutagenesis in vivo, we simply inject Xenopus eggs with the appropriate mRNA and suppressor tRNA.
Probing receptor function with fluorescence
Summary of dissertation work
We have identified side chains that are important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other using a FRET assay.
In vivo incorporation of unnatural amino acids into ion channels in the Xenopus oocyte expression system. An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.
Dissecting the functions of conserved prolines within transmembrane
- Introduction
- Results
- Experimental approach
- Strategy and general observations
- Discussion
- Conclusions
- Experimental
- Molecular biology
- Oocyte preparation and RNA injection
- Electrophysiology
- References
Transmembrane proline residues are a characteristic feature of GPCRs and are found in five of the seven D2 receptor TMs. Probing the role of cation-pi interaction at GPCR binding sites using unnatural amino acids.
Probing side chain conformations and rearrangements in receptor
Introduction
- GPCR activation
- nAChR activation
- Probing side chain conformation by mutagenesis
Interestingly, another “switch” was observed a helical turn below W6.48 in crystal structures of the β2-adrenergic receptor. The tolerance of this significant structural and electrostatic perturbation helps inform the location of the W6.48 side chain within GPCR binding sites.
Results and Discussion
- Mutagenesis probing conformational rearrangements of C6.47 upon
- Investigating the role of a F6.44/I3.40 switch in activation of the D2
- Synthesis and use of 5-aminomethyltryptophan to probe the W6.48
- Synthesis, mutagenesis, and modeling of β-methyltryptophan and β-
- Synthesis
- Mutagenesis
- Conformational effects
- Double mutant cycle analysis to probe steric effects of β-
In particular, we were interested in probing W6.48 in the M2 muscarinic acetylcholine receptor and in the D2 dopamine receptor. In the speculative case that the D2 W6.48[5-NH2CH2Trp] mutant expresses but is non-functional, an interesting contrast with the M2 receptor emerges. This could explain the surprisingly good tolerance of this side chain in the M2 receptor.
Conclusions
Overall, the lack of predictability suggests that our model of the active receptor binding site is incorrect, or that the mutations designed to simply alter sterics have unforeseen consequences. At this point it is difficult to know the true energetic consequences of a mutation in the actual receptor. Steric and electrostatic effects of the local protein environment certainly complicate the simple conformational analysis applied to Thr/aThr or Ile/aIle or the more sophisticated χ1, χ2 energy analysis applied to the β-methyl analogues in this chapter.
Experimental
- Molecular biology
- Microinjection
- Electrophysiology
- Energy calculations
- Synthesis
Trifluoroacetic acid was added (1 ml) and the reaction was stirred under argon for 1 hour to obtain the reaction solution. Aqueous 1M NaOH was added (0.5 ml) and the reaction was stirred at room temperature for 20 hours. Triethylamine (24 µl, 0.17 mmol, 3 eq) was added and the reaction was stirred at room temperature for 6 hours.
Mutation of a single TMVI residue, Phe282, in the β2-adrenergic receptor leads to structurally distinct activated receptor conformations. Asymmetric-synthesis of unusual amino acids - synthesis of optically pure isomers of N-indole-(2-mesitylenesulfonyl)-Beta-methyltryptophan. This chapter is adapted from research previously published in the Journal of Biological Chemistry: Blum, A.
Binding interactions to the complementary subunit of the α4β4 receptor
- Introduction
- Results
- General strategy
- Mutagenesis studies of the Leu NH
- Mutagenesis studies of the Asn CO
- Discussion
- Experimental
- Mutagenesis
- Microinjection
- Electrophysiology
- References
Mutagenesis of the backbone NH (γL119/δL121 mutations) and backbone CO (γV108/δV110 mutations) of the muscle-type nAChR. The cationic N binds to the major component of the agonist binding site in the α subunit, and the hydrogen bond acceptor binds to the complementary, non-α subunit. Nevertheless, the studies of the α4β2 receptor provide support for the idea that mutations of the Leu NH disrupt a hydrogen bond to the agonist.
An unusual pattern of ligand-receptor interactions for the α7 nicotinic
Introduction
This complementary side of the binding site is thought to recognize the hydrogen bond acceptor part of the classic nicotine pharmacophore. Several side chains on the complementary side of the binding site are also positioned to form potential ligand contacts. In the present work, we use unnatural amino acid mutagenesis to assess the binding of the fully effective and moderately potent agonist varenicline to the aromatic box residues of the main face of the α7 receptor.
Results
- Experimental design
- Unnatural amino acid mutagenesis to probe cation-π interactions
- Varenicline interactions with the binding site’s principal face
- Probing the canonical hydrogen bond of the nicotinic
ACh and epibatidine, varenicline does not form a cation-π interaction with TrpB. the observation further supports a unique binding mode in the aromatic box for agonists at α7. To determine whether varenicline forms a cation-π interaction to one or more aromatic residues in the main face of the binding site, we incorporated unnatural aromatic amino acid analogs with attenuated cation-π binding ability and investigated for a concomitant reduction in function of the receptor (Figure 5.3B). The remaining aromatic residues of the main site, TyrA, TyrC1, and TyrC2, were also probed for cation-π binding to varenicline.
Discussion
- Cation-π interactions to the “aromatic box” residues of the principal
- Hydrogen bonding and steric effects on the principal face
- Hydrogen bonding to the complementary face
- Interactions with complementary face side chains
We find that, in the α7 receptor, ACh and epibatidine are largely insensitive to the V108Vah mutation that weakens the hydrogen bond acceptor capacity of the Asn107 CO, with EC50 shifts < 2-fold (Table 5.2). Taken together, it is possible that the α7 receptor either engages the agonist hydrogen bond acceptor with other groups or does not have energetically significant contacts with it. Regarding the possibility that this hydrogen bond acceptor group does not form functionally important receptor contacts, it is worth remembering.
Conclusions
EC50, but increases the relative efficacy of varenicline from 15% to 125% in the α4β2 receptor, the intended target of this drug.13 The substantial increase in EC50 we observe for W55A in the α7 receptor represents a dramatically different phenotype and indicates that this residue contributes differentially to receptor function for α7 versus α4β2.
Experimental
- Molecular biology
- Microinjection
- Electrophysiology
The amber suppressor tRNA THG7336 was used for nonsense suppression at all sites except Val108 and Leu119, for which the opal suppressor TQOpS'37,38 was used. Stage V-VI Xenopus laevis oocytes were harvested and injected with RNAs as previously described. 5–25 ng of α7 mRNA was co-injected with ~20 ng of RIC-3 mRNA per oocyte. In the case of low maximum currents sometimes observed in nonsense suppression experiments, presumably due to low expression, a second RNA injection was necessary 24 h after the first injection.
Creating an alpha7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses. Structural characterization of binding mode of smoking cessation agents to nicotinic acetylcholine receptors by study of ligand complexes with acetylcholine binding protein. Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper.
Efforts toward single molecule fluorescence imaging of nAChRs
Introduction
Previously, the laboratories of Dougherty and Lester reported the successful integration of a BODIPY fluorophore into the nAChR by nonsense suppression in Xenopus oocytes and single-molecule imaging of these fluorescent receptors.9 The fluorophore used was BODIPYFL, which was conjugated to a lysine side chain. Figure 6.1A). This side chain was incorporated into the β19' site of the muscle-type nAChR and the presence of functional receptors on the cell membrane was confirmed by electrophysiology. A logical extension of this work is the integration of two different fluorophores site-specifically into the nAChR to enable Förster resonance energy transfer (FRET) studies.
Results and Discussion
- Background fluorescence from tRNA-BODIPY
- Optimization of vitelline membrane removal
- Two-color imaging and single puncta analysis
- Attempts at fluorophore conjugation by azide-alkyne
- Design and synthesis of a fluorescence-quenched tRNA
To assess whether the nature of the BODIPY moiety added to alter the background signal, we also examined fluorescence from cells injected with. In the lower panel, the cell was injected with β-subunit GFP-tagged nAChR mRNA. Peak whole-cell currents from cells injected with mixtures of 10 ng β70TAG nAChR mRNA and 10 ng of the specified tRNA.
Conclusions
Experimental
- Molecular biology and in vivo expression
- Electrophysiology
- Oocyte membrane preparation
- Total internal reflection fluorescence (TIRF) imaging
- Azido dye labeling under copper-catalyzed click conditions
- Two-color TIRF imaging and image analysis
- tRNA synthesis and characterization
In another alternative condition20, complete digestion of the vitelline membrane is achieved by protease treatment (0.05 mg/ml in ND96, Type VIII, Sigma) with gentle shaking for 30 min. For these experiments, an Optosplit II (Cairn Research) beam splitter was installed in the fluorescence emission light path directly in front of the camera. In our experiments, the intensity of the donor channel was significantly higher than the intensity of the acceptor channel, so a neutral density filter was placed in the path of the donor channel.
The gel was imaged on a Typhoon FLA 9000 imager (GE Healthcare Life Sciences) both at 473 nm excitation with a 515–545 nm bandpass emission filter and at 532 nm excitation with a 560–580 nm bandpass emission filter. Incorporation of site-specific biotinylated amino acids to identify surface-exposed residues in integral membrane proteins. Role of the 5'-terminal phosphate of tRNA for its function during the elongation cycle of protein biosynthesis.
Attempts to develop a FRET assay for the GPCR-G protein interaction
- Introduction
- Results and Discussion
- Construction of fluorescent protein fusions
- Tests of construct function in vivo
- Formation and fluorescence imaging of plasma membrane sheets 188
- Conclusions
- Experimental
- Molecular biology and in vivo expression
- Electrophysiology
- Membrane preparation
- FLIM imaging
- References
This is the state captured in the β2AR-Gs crystal structure; apyrase digestion was necessary to form a stable receptor-G protein complex for crystallography.1 In a cellular context, this would be a transient intermediate: GTP then binds, causing dissociation of the G protein from the receptor and the Gα subunit from Gβγ is induced (Figure 7.1). An ideal assay for structure-function studies will allow unnatural amino acid mutagenesis of the receptor and of the G protein. FRET between fluorescent protein (FP) fusions of the receptor and the G protein could provide a readout of the dissociation of the complex.
