Amber Codon Suppression Research Articles

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5′-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5′-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3′-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

Conformational Dynamics of the Intrinsic Ligand in the CNBHD of the Voltage-Gated Potassium Channel hERG

Human ether-á-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. Characteristic slow closing (deactivation) in hERG is regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). An intrinsic ligand in hERG is located at the PAS-CNBHD interface, but its mechanism is not well understood. Our recent work with co-expressed channel fragments in which the hERG PAS domain fused to CFP was expressed in trans with the ‘core’ of the hERG channel (i.e. a channel lacking the PAS domain that is fused to Citrine at the C-terminus, hERG ΔPAS-Citrine) showed that deactivation gating was disrupted (was made faster) by intrinsic ligand mutations and that the activation time course of the channel was perturbed (was made faster), indicating that the intrinsic ligand is necessary for the functional and structural interaction of the PAS domain and the CNBHD. To better understand the dynamics for intrinsic ligand regulation of hERG gating, we incorporated the fluorescent non-canonical amino acid ANAP using amber codon suppression at the intrinsic ligand, and engineered dihistidines that were structurally adjacent to this ANAP, to allow us to probe a change (1) using an environmentally-dependent spectral shift of ANAP and/or (2) using transition metal FRET. This technique will determine how the intrinsic ligand at the PAS-CNBHD interface may undergo conformational changes with voltage to regulate channel deactivation gating.

Incorporation of Modified Amino Acids by Engineered Elongation Factors with Expanded Substrate Capabilities.

Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu’s substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair.

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.

Tag-on-Demand: exploiting amber codon suppression technology for the enrichment of high-expressing membrane protein cell lines.

Membrane proteins play key roles in the evolution of numerous diseases and as a result have become the most dominant class of targets for therapeutic intervention. However, their poor expression and detection oftentimes prohibit drug discovery and screening efforts. Herein, we have developed an approach, named 'Tag-on-Demand' that exploits amber suppression to control the expression of 'tagged' membrane proteins for detection and selections, yet can be turned off for expression of the protein in its native form. Utilizing an engineered Chinese hamster ovary cell line capable of efficient amber suppression, we evaluated the expression of a diverse panel of model membrane proteins and demonstrated the enrichment of cells with improved expression profiles, where ~200-800% improvement in total protein expression levels were observed over pre-sorted populations after a single round of fluorescence-activated cell sorting. Furthermore, results were most striking for the typically difficult-to-express G protein-coupled receptor, CXCR2, where ~2.5-fold improvement in surface expression was observed. We anticipate that the Tag-on-Demand approach will be suitable not only for membrane protein cell line development but also for the development of intracellular and secreted protein cell lines in expression systems for which amber suppression technology exists, including bacterial, yeast, insect and cell-free expression systems.

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine.

The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-L-phenylalanine (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO2F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4122 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO2F or Asn149pNO2F sfGFP with wild-type sfGFP resulted in small root-mean-square deviations, illustrating that these residues do not significantly alter the protein structure and supporting the use of pNO2F as an effective spectroscopic reporter of local protein structure and dynamics.

Visualizing conformational dynamics of proteins in solution and at the cell membrane.

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers.

Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E.coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R.sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E.coli. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein via mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in R.sphaeroides expands research capabilities in the photosynthetic field.

Probing Local Protein Environments with a Vibrational Reporter Unnatural Amino Acid

Vibrational reporter unnatural amino acids have the potential to serve as sensitive, site-specific probes of local protein environments. Here the vibrational reporter unnatural amino acid 4-cyano-L-phenylalanine (pCNF) was utilized to probe multiple distinct sites in the Heme Nitric Oxide and/or Oxygen (H-NOX) binding domain from Caldanaerobacter subterraneus. pCNF is an effective reporter of local environment due to the position and sensitivity of the nitrile stretch frequency. pCNF was genetically incorporated into H-NOX into three distinct sites using the amber codon suppression methodology. The sites were selected to represent a myriad of local protein environments. The frequency of the nitrile symmetric stretch of pCNF and the temperature dependence of this frequency was utilized to assess the solvation state of the nitrile group in each of these sites in the protein. This analysis was aided by measuring the frequency and temperature dependence of the nitrile stretch vibration of pCNF in a variety of solvents to mimic different protein environments. These results probing the local environments in H-NOX will be presented.

Artificial Modification of Histones and the Effect on Epigenetics

In order for cells to package all of their genetic material, DNA is wrapped around eight histone proteins (two copies each of H2A, H2B, H3, and H4) to make up the nucleosome. Nucleosomes are the fundamental building blocks of chromatin, which is the first step of compaction and the first level of gene regulation. The phrase “histone code” was coined in regard to those molecular processes that regulate the eukaryotic genome. Further study of the “histone code” and epigenetics has revolutionized the scientific community's understanding of the regulation of gene expression. The Histone H4 N-terminal tail has a region called the basic patch. It is thought that this region is a hub for histone-modifying activity. Histone H2B lysine-123 (H2BK123) is monoubiquitinated and this modification is required for the proper regulation of transcription. In this study, H2BK123ub levels were measured and preparations were made to develop a novel amber codon suppression screen to study the histone H4 basic patch. The overarching goal of the study was to artificially modify histones and their post-translational modification processes in order to understand their mechanisms and therefore better understand epigenetics.

Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Non-canonical Amino Acids into Photosynthetic Reaction Centers

Bacterial reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. They capture solar energy in a highly efficient combination of energy and electron transfer steps. RCs possess two branches of chromophores arranged in nearly identical paths, L and M. Following photoexcitation, however, electrons are only transferred down the L branch of chromophores. Despite intense effort, the origin(s) of this unidirectional electron transfer is not fully understood, and even the role of intermediary electron acceptors, is unclear. To better study these issues, we have recently transferred an amber codon suppression system from E. coli into the bacteria needed to produce RCs, R. sphaeroides. Numerous challenges were overcome to implement amber suppression in R. sphaeroides; they further inform on the often-unforeseeable hurdles that can be involved in host transfer of amber suppression from E. coli. With this system in hand, we can more finely dissect the role of key amino acids in a way previously inaccessible to photosynthetic research using non-canonical amino acids. Initial work focuses on the -OH dipole strength in tyrosine M210, believed to play a key role in modulating primary charge separation. This is accomplished by using differently halogenated tyrosine variants (e.g. mono-Cl, -Br and -I) to inspect the gradual effect of these structural changes on RC function. Detailed characterization of RC mutants incorporating these variants will be described. Non-canonical amino acids in the form of site-specific probes are also being pursued. These tools greatly broaden the scope of site-directed mutagenesis in this complex membrane protein. Their additional insight may help us better understand protein design in this model for electron transfer.

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E.coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:β2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122β ↔ [W48β?] ↔ Y356β ↔ Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:β2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns-μs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s-1 when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ∼3-5 d, whereas a whole directed evolution can be performed in 3-10 rounds, depending on selection efficiency.

Mapping the Binding Site for Escitalopram and Paroxetine in the Human Serotonin Transporter Using Genetically Encoded Photo-Cross-Linkers.

In spite of the important role of the human serotonin transporter (hSERT) in depression treatment, the molecular details of how antidepressant drugs bind are still not completely understood, in particular those related to potential high- and low-affinity binding sites in hSERT. Here, we utilize amber codon suppression in hSERT to encode the photo-cross-linking unnatural amino acid p-azido-l-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced cross-linking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective serotonin reuptake inhibitors (SSRIs). We find that the two antidepressant drugs exclusively cross-link to azF incorporated at the high-affinity binding site of hSERT, while cross-linking is not observed at the low-affinity binding site. Combined with previous homology models and recent structural data on hSERT, our results provide important information to understand the molecular details of these clinical relevant binding sites.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking.

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein–protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein–protein interactions and their interfaces in living cells.

Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids.

Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.

Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.

Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT.

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

We have previously shown that a histidine (H420) in the first intracellular helix (A’ helix) of cyclic nucleotide-gated (CNGA1) channels is involved in potentiation of CNGA1 currents by transition metal ions. The binding of transition metals stabilizes the open state of the channel, so understanding the details of metal ion coordination will provide insight into the structural changes that occur after ligand binding to promote channel opening. Because structural studies suggest that the A’ helix is proximal to the membrane, we tested the hypothesis that a metal ion is coordinated between H420 and the intracellular leaflet of the bilayer with transition metal FRET (tmFRET) and patch-clamp fluorometry. To label CNGA1 channels, the amber codon suppression strategy was used to incorporate a fluorescent, noncanonical amino acid, L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) near H420. We chose Co2+ (1-30 μM) to potentiate CNGA1 currents and serve as a FRET acceptor for L-Anap in inside-out patches from Xenopus oocytes. We found that Co2+ quenched Anap fluorescence by ∼50%, and the H420Q mutation significantly attenuated quenching, providing further evidence that Co2+ binds to H420. Next, to test whether the membrane provides a second Co2+ ligand, we added high-affinity metal binding sites to the membrane using a novel, metal-chelating fatty acid (C18-NTA). We found that metal potentiation was increased for low (1-3 μM) Co2+ concentrations with C18-NTA-Co2+, and this effect required H420. These results indicate that membrane-bound metal ions interact with the A’ helix, suggesting that the mechanism for metal potentiation is metal ion coordination between H420 and a lipid with an anionic headgroup with lower affinity for Co2+ compared to C18-NTA. This arrangement of the A’ helix relative to the membrane potentiates channel opening and may be an important structural transition that occurs with cyclic nucleotide regulation.