
Binding of the full agonist cAMP induces large structural changes and favorable energetics in the C-terminal region of the cyclic nucleotide-gated ion channel SthK, while the partial agonist cGMP promotes a smaller fraction of the active conformation.


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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.

Significance Statement Allosteric regulation is pivotal for the function of most proteins, especially ion channels like the cyclic nucleotide-binding domain (CNBD) channels. This study examines the allosteric mechanism of ligand binding in the C-terminal region of the prokaryotic CNBD ion channel SthK using steady-state and time-resolved tmFRET. We uncovered significant structural and energetic changes induced by ligand binding with the full-agonist cAMP and the weak partial agonist cGMP. Our approach also highlights the effectiveness of using fluorescence lifetimes to reveal conformational heterogeneity and free energy changes in proteins. These findings deepen our understanding of CNBD channel activation overall and lay the groundwork for a more comprehensive characterization of the effects of mutations and pharmacological agents in these channels.

### Competing Interest Statement

The authors have declared no competing interest.

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

structural-and-energetic-insights-into-ligand-induced-conformational-changes-in-a-cyclic-nucleotide-gated-ion-channel-using-time-resolved-transition-metal-ion-fret


Structural and Energetic Insights into Ligand-Induced Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel using Time-Resolved Transition Metal Ion FRET


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Newly developed techniques of rapid temperature jumps (Tjumps) and sustained temperature steps (Tsteps) provide a powerful tool to study the temperature dependence of ion channel function and gating mechanisms.


Perturbing the temperature of a system modifies its energy landscape thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage-clamp, while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 10 ºC in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with submillisecond time resolution by measuring the time-course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir 1.1b, which reveals a highly temperature-sensitive blockage relief and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane proteins thermodynamics.

### Competing Interest Statement

The authors have declared no competing interest.

ION CHANNEL THERMODYNAMICS STUDIED WITH TEMPERATURE JUMPS MEASURED AT THE CELL MEMBRANE

rapid-temperature-jumps-and-steps-reveal-insights-into-ion-channel-thermodynamics


Rapid Temperature Jumps and Steps Reveal Insights into Ion Channel Thermodynamics



Development of ElegansBot for replicating C. elegans locomotion behaviors.


Locomotion is a fundamental behavior of Caenorhabditis elegans ( C. elegans ). Previous works on kinetic simulations of animals helped researchers understand the physical mechanisms of locomotion and the muscle-controlling principles of neuronal circuits as an actuator part. It has yet to be understood how C. elegans utilizes the frictional forces caused by the tension of its muscles to perform sequenced locomotive behaviors. Here, we present a two-dimensional rigid body chain model for the locomotion of C. elegans by developing Newtonian equations of motion for each body segment of C. elegans . Having accounted for friction-coefficients of the surrounding environment, elastic constants of C. elegans , and its kymogram from experiments, our kinetic model (ElegansBot) reproduced various locomotion of C. elegans such as, but not limited to, forward-backward-(omega turn)-forward locomotion constituting escaping behavior and delta-turn navigation. Additionally, ElegansBot precisely quantified the forces acting on each body segment of C. elegans to allow investigation of the force distribution. This model will facilitate our understanding of the detailed mechanism of various locomotive behaviors at any given friction-coefficients of the surrounding environment. Furthermore, as the model ensures the performance of realistic behavior, it can be used to research actuator-controller interaction between muscles and neuronal circuits.

### Competing Interest Statement

The authors have declared no competing interest.

ElegansBot: Development of equation of motion deciphering locomotion including omega turns of Caenorhabditis elegans

elegansbot-understanding-c-elegans-locomotion


ElegansBot: Understanding C. elegans Locomotion



Proposing CONFDIFF, a force-guided SE(3) diffusion model for efficient protein conformation generation.


protein-conformation-generation-via-force-guided-se-3-diffusion-models-enhancing-protein-sampling-efficiency


Protein Conformation Generation via Force-Guided SE(3) Diffusion Models: Enhancing Protein Sampling Efficiency



Ion gradients enable rapid cellular response to environmental perturbations through cytoskeleton-mediated information transmission.


modeling-ion-based-information-dynamics-in-cells-using-cell-reservoir-computing


Modeling Ion-Based Information Dynamics in Cells Using Cell-Reservoir Computing



The author demonstrates that fine-tuning a general purpose large language model can enhance the analysis and design of DNA sequences by leveraging chain-of-thought responses and expert chaining.


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Learning DNA Structural Biophysics with LLMs



The author explores the structural elements contributing to mechanosensitivity in OSCA channels, highlighting the role of the amphipathic helix and lipid-interacting residues in response to mechanical stimuli.


The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension[1][1]. Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e., they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). In an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization[2][2]. Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

### Competing Interest Statement

The authors have declared no competing interest.

 [1]: #ref-1
 [2]: #ref-2

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli

Structure-guided-Mutagenesis-of-OSCAs-Reveals-Mechanosensitive-Activation


Structure-guided Mutagenesis of OSCAs Reveals Mechanosensitive Activation
