Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

Gordon, Sharona E.; Evans, Eric G.B.; Otto, Shauna C.; Tessmer, Maxx H.; Shaffer, Kyle D.; Gordon, Moshe T.; Petersson, E. James; Stoll, Stefan; Zagotta, William N.

doi:10.1016/j.bpj.2024.01.034

Cited by 6 publications

(5 citation statements)

References 31 publications

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“…Thus, with Acd as a donor, these acceptors allowed us to measure both short (10-20 Å) and long distances (25-50 Å) across SthK C-term (Fig. 2B) (19).…”

Section: Resultsmentioning

confidence: 99%

“…Adenosine 3’,5’-cyclic monophosphate sodium salt monohydrate (cAMP) and guanosine 3’,5’-cyclic monophosphate sodium salt (cGMP) were purchased from Sigma Aldrich and both prepared at 16 mM in 150 mM KCl, 50 mM Tris and 10% glycerol, pH 7.4. Metal acceptor complexes, [Fe(phenM) 3 ] 2+ , [Ru(bpy) 2 phenM] 2+ , and [Cu(TETAC)] 2+ for tmFRET were prepared as previously described (19, 20).…”

Section: Methodsmentioning

confidence: 99%

“…Protein was confirmed as tetrameric by analytical SEC and diluted 1:2 to 1:5 into KBT into quartz cuvettes. Acd fluorescence intensity was measured at 10 second intervals using Jobin Yvon Horiba FluoroMax-3 spectrofluorometer in anti-photobleaching mode (Edison, NJ) as described previously (18, 19). Acceptor molecules at final concentrations of 6 µM [Fe(phenM) 3 ] 2+ , 20 µM [Ru(bpy) 2 phenM] 2+ or 10 µM [Cu(TETAC)] 2+ were added to the cuvettes followed by 160 μM final concentration of either cAMP or cGMP.…”

Section: Methodsmentioning

confidence: 99%

“…The measured donor-only lifetimes (in KBT and High-KBT) were single exponential, and the time constant was previously measured and held fixed for the global fitting. The acceptor complex R0 values were determined previously (19) and held constant at 41.8 Å and 43.5 Å for [Fe(phenM)3] 2+ and [Ru(bpy)2phenM] 2+ respectively 19 . For the single Gaussian lifetime fits, the fraction donor only was held constant within experiments from the same labeled protein samples.…”

Section: Mass Photometrymentioning

confidence: 99%

“…In this study, we utilized our recently described steady-state and time-resolved tmFRET methods to characterize the ligand-induced structural changes and energetics in the isolated Cterminal fragment of SthK (18)(19)(20). In tmFRET, a donor fluorophore is paired with a transition metal ion, such as Cu 2+ , Fe 2+ , or Ru 2+ , as an acceptor.…”

Section: Introductionmentioning

confidence: 99%

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Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET

Eggan,

Gordon,

Zagotta

2024

Preprint

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Cyclic nucleotide-binding domain (CNBD) ion channels play crucial roles in cellular-signaling and excitability and are regulated by the direct binding of cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP). However, the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood. The prokaryotic CNBD channel SthK offers a valuable model for investigating this allosteric mechanism. In this study, we investigated the conformational dynamics and energetics of the SthK C-terminal region using a combination of steady-state and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) experiments. We engineered donor-acceptor pairs at specific sites within a SthK C-terminal fragment by incorporating a fluorescent noncanonical amino acid donor and metal ion acceptors. Measuring tmFRET with fluorescence lifetimes, we determined intramolecular distance distributions in the absence and presence of cAMP or cGMP. The probability distributions between conformational states without and with ligand were used to calculate the changes in free energy (ΔG) and differences in free energy change (ΔΔG) in the context of a simple four-state model. Our findings reveal that cAMP binding produces large structural changes, with a very favorable ΔΔG. In contrast to cAMP, cGMP behaved as a partial agonist and only weakly promoted the active state. Furthermore, we assessed the impact of protein oligomerization and ionic strength on the structure and energetics of the conformational states. This study demonstrates the effectiveness of time-resolved tmFRET in determining the conformational states and the ligand-dependent energetics of the SthK C-terminal region.

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“…Thus, with Acd as a donor, these acceptors allowed us to measure both short (10-20 Å) and long distances (25-50 Å) across SthK C-term (Fig. 2B) (19).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mass Photometrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET

Eggan,

Gordon,

Zagotta

2024

Preprint

Self Cite

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Improved Large-Scale Synthesis of Acridonylalanine for Diverse Peptide and Protein Applications

Marmorstein,

Pagar,

Hummingbird

et al. 2024

Bioconjugate Chem.

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Fluorescent unnatural amino acids give biochemists, biophysicists, and bioengineers new ways to probe the properties of proteins and peptides. Here, the synthesis of acridon-2-ylalanine (Acd) is optimized for large-scale production to enable ribosomal incorporation through genetic code expansion (GCE), and fluorenylmethoxycarbonyl (Fmoc)-protected Acd is prepared for solid-phase peptide synthesis (SPPS). We demonstrate the utility of Acd in several applications: first, Acd quenching by Tyr is used in the design of fluorescent protease sensors made by SPPS. Second, we demonstrate Acd incorporation into a lanthanide-binding peptide that is generated either by GCE or by SPPS and demonstrate the utility of Acd for sensitizing the emission of Eu 3+ . Finally, Acd is inserted into the intrinsically disordered protein, α-synuclein, using GCE and used to study ion binding and aggregation.

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Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET

Eggan,

Gordon,

Zagotta

2024

eLife

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Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

Cited by 6 publications

References 31 publications

Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET

Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET

Improved Large-Scale Synthesis of Acridonylalanine for Diverse Peptide and Protein Applications

Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET

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