Sharona E. Gordon

Transition-metal ion Förster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor– acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Förster distance (R ₀ ) can be tuned without changing protein labeling. Cu²⁺, Cu²⁺ complexed with nitrilotriacetic acid (NTA), and Fe²⁺ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]²⁺, [CuNTA(bpyA)]²⁺, and [Fe(bpyA)]²⁺ acceptors provided R ₀ values ranging from 13.9 to 21.2 Å. Analysis of time-resolved tmFRET data recovered donor–acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]²⁺ did not appreciably resolve the distance increase, whereas adding NTA shifted R ₀ toward shorter distances and enabled detection of an increase from 12.1 to 14.4 Å. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.

Outer mitochondrial membrane fusion is mediated by the mitofusin paralogs Mfn1 and Mfn2. Nucleotide-driven self-assembly and conformational changes are required for regulated membrane fusion activity, but the allosteric mechanisms remain enigmatic due to incomplete structural information. In this study, we investigate the GTP-coupled conformational dynamics of Mfn1 using time-resolved transition metal ion fluorescence resonance energy transfer (tmFRET). Using the minimal Mfn1 construct with the GTPase domain and helical bundle 1 (HB1) connected by Hinge 2, we engineered FRET pairs by incorporating a fluorescent noncanonical amino acid donor and a metal ion acceptor. For each state of the catalytic cycle, we measured tmFRET with fluorescence lifetimes and determined distance distributions, which can capture complex structural heterogeneity. Our distance measurements for the GDP-bound state matched predictions from the atomic resolution structure, establishing that the same open state, with GTPase and HB1 domains far apart, exists in solution. Our data reveal that the transition state is not a single closed state with HB1 stably contacting the GTPase domain. Rather, the distance distributions indicate that the presence of GDP + Pi results in an equilibrium between the open and closed states. We also captured the GTP-bound and nucleotide-free states of Mfn1. GTP binding favors the open state, and the conformation of the apo state is distinct from any nucleotide-bound state. Together, these findings redefine our understanding of GTP-driven conformational dynamics in Mfn1, demonstrating an unexpected conformational reversal in a single catalytic cycle and a heterogeneous transition state ensemble with implications for the mechanism and regulation of mitochondrial membrane fusion.

The human voltage-gated proton channel (hH _v 1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hH _v 1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands, such as Zn ²⁺ . Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hH _v 1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hH _v 1 purified from E. coli . We used genetic code expansion (GCE) to generate a library of 14 full-length hH _v 1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hH _v 1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hH _v 1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn ²⁺ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

The human voltage-gated proton channel (hHv1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hHv1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands such as Zn2+. Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hHv1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hHv1 purified from E. coli. We used genetic code expansion (GCE) to generate a library of 14 full-length hHv1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hHv1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hHv1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn2+ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

The human voltage-gated proton channel (hH _v 1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hH _v 1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands such as Zn ²⁺ . Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hH _v 1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hH _v 1 purified from E. coli . We used genetic code expansion (GCE) to generate a library of 14 full-length hH _v 1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hH _v 1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hH _v 1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn ²⁺ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

Cyclic nucleotide-binding domain (CNBD) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describe conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNBD channel SthK has been a useful model for better understanding these allosteric mechanisms. Previously, we applied time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the CNBD of a soluble C-terminal fragment of the SthK protein, SthK_Cterm (Eggan et al., 2024). Here, we used a similar strategy to measure the conformational energetics of the CNBD in the full-length channel, SthK_Full, and compared them to measurements from SthK_Cterm. We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time-correlated single-photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthK_Cterm and SthK_Full. Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNBD channels specifically.

Cyclic nucleotide-binding domain (CNBD) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine-or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describe conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNBD channel SthK has been a useful model for better understanding these allosteric mechanisms. Here, we applied time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the CNBD of SthK in both a soluble C-terminal fragment of the protein, SthKCterm, and in the full-length channel, SthKFull. We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time correlated single photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthKCterm and SthKFull. Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNBD channels specifically.

Cyclic nucleotide-binding domain (CNBD) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine-or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describe conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNBD channel SthK has been a useful model for better understanding these allosteric mechanisms. Here, we applied time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the CNBD of SthK in both a soluble C-terminal fragment of the protein, SthK _Cterm , and in the full-length channel, SthK _Full . We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time correlated single photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthK _Cterm and SthK _Full . Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNBD channels specifically.

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.

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.