Lifetime of ground conformational state determines the activity of structured RNA
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Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
The phosphatase and tensin homolog (PTEN) is a vital protein that maintains an inhibitory brake for cellular proliferation and growth. Accordingly, PTEN loss-of-function mutations are associated with a broad spectrum of human pathologies. Despite its importance, there is currently no method to directly monitor PTEN activity with cellular specificity within intact biological systems. Here we describe the development of a FRET-based biosensor using PTEN conformation as a proxy for the PTEN activity state, for two-photon fluorescence lifetime imaging microscopy. We identify a point mutation that allows the monitoring of PTEN activity with minimal interference to endogenous PTEN signaling. We demonstrate imaging of PTEN activity in cell lines, intact Caenorhabditis elegans and in the mouse brain. Finally, we develop a red-shifted sensor variant that allows us to identify cell-type-specific PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.
TCR catch bonds nonlinearly control CD8 cooperation to shape T cell specificity
Naturally evolved T-cell receptors (TCRs) exhibit remarkably high specificity in discriminating non-self antigens from self-antigens under dynamic biomechanical modulation. In contrast, engineered high-affinity TCRs often lose this specificity, leading to cross-reactivity with self-antigens and off-target toxicity. The underlying mechanism for this difference remains unclear. Our study reveals that natural TCRs exploit mechanical force to form optimal catch bonds with their cognate antigens. This process relies on a mechanically flexible TCR–pMHC binding interface, which enables force-enhanced CD8 coreceptor binding to MHC-α1α2 domains through sequential conformational changes induced by force in both the MHC and CD8. Conversely, engineered high-affinity TCRs create rigid, tightly bound interfaces with cognate pMHCs of their parental TCRs. This rigidity prevents the force-induced conformational changes necessary for optimal catch-bond formation. Paradoxically, these high-affinity TCRs can form moderate catch bonds with non-stimulatory pMHCs of their parental TCRs, leading to off-target cross-reactivity and reduced specificity. We have also developed comprehensive force-dependent TCR–pMHC kinetics-function maps capable of distinguishing functional and non-functional TCR–pMHC pairs and identifying toxic, cross-reactive TCRs. These findings elucidate the mechano-chemical basis of the specificity of natural TCRs and highlight the critical role of CD8 in targeting cognate antigens. This work provides valuable insights for engineering TCRs with enhanced specificity and potency against non-self antigens, particularly for applications in cancer immunotherapy and infectious disease treatment, while minimizing the risk of self-antigen cross-reactivity.
AFsample2 predicts multiple conformations and ensembles with AlphaFold2
Understanding protein dynamics and conformational states is crucial for insights into biological processes and disease mechanisms, which can aid drug development. Recently, several methods have been devised to broaden the conformational predictions made by AlphaFold2 (AF2). We introduce AFsample2, a method using random MSA column masking to reduce co-evolutionary signals, enhancing structural diversity in AF2-generated models. AFsample2 effectively predicts alternative states for various proteins, producing high-quality end states and diverse conformational ensembles. In the OC23 dataset, alternate state models improved (ΔTM>0.05) in 9 out of 23 cases without affecting preferred state generation. Similar results were seen in 16 membrane protein transporters, with 11 out of 16 targets showing improvement. TM-score improvements to experimental end states were substantial, sometimes exceeding 50%, improving from 0.58 to 0.98. Additionally, AFsample2 increased the diversity of intermediate conformations by 70% compared to standard AF2, producing highly confident models potentially representing intermediate states. For four targets, predicted intermediate states were structurally similar to known structural homologs in the PDB, suggesting that they are true intermediate states. These findings indicate that AFsample2 can used to provide structural insights into proteins with multiple states, as well as potential paths between the states.
Collective quantum enhancement in critical quantum sensing
Critical systems represent a valuable resource in quantum sensing and metrology. Critical quantum sensing (CQS) protocols can be realized using finite-component phase transitions, where criticality arises from the rescaling of system parameters rather than the thermodynamic limit. Here, we show that a collective quantum advantage can be achieved in a multipartite CQS protocol using a chain of parametrically coupled critical resonators in the weak-nonlinearity limit. We derive analytical solutions for the low-energy spectrum of this unconventional quantum many-body system, which is composed of locally critical elements. We then assess the scaling of the quantum Fisher information with respect to fundamental resources. We demonstrate that the coupled chain outperforms an equivalent ensemble of independent critical sensors, achieving quadratic scaling in the number of resonators. Finally, we show that even with finite Kerr nonlinearity or Markovian dissipation, the critical chain retains its advantage, making it relevant for implementing quantum sensors with current microwave superconducting technologies.
Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1
Mammalian sodium-coupled high-affinity choline transporter CHT1 uptakes choline in cholinergic neurons for acetylcholine synthesis and plays a critical role in cholinergic neurotransmission. Here, we present the high-resolution cryo-EM structures of human CHT1 in apo, substrate- and ion-bound, hemicholinium-3-inhibited, and ML352-inhibited states. These structures represent three distinct conformational states, elucidating the structural basis of the CHT1-mediated choline uptake mechanism. Three ion-binding sites, two for Na+ and one for Cl–, are unambiguously defined in the structures, demonstrating that both ions are indispensable cofactors for high-affinity choline-binding and are likely transported together with the substrate in a 2:1:1 stoichiometry. The two inhibitor-bound CHT1 structures reveal two distinct inhibitory mechanisms and provide a potential structural platform for designing therapeutic drugs to manipulate cholinergic neuron activity. Combined with the functional analysis, this study provides a comprehensive view of the structural mechanisms underlying substrate specificity, substrate/ion co-transport, and drug inhibition of a physiologically important symporter.
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