A comparative biophysical analysis of glucose translocation using both the native Cryo-EM structure and the AI-predicted AlphaFold model is conducted, establishing that rigid AI-generated architectures lack the essential internal free volume and conformational plasticity required to sustain accurate thermodynamic profiling of dynamic membrane transporters.
Abstract
The facilitative transport of hexose through the GLUcose Transporter type 4 (GLUT4) is essential for cellular metabolism and is regulated by allosteric nucleotide interactions. In this study, we conducted a comparative biophysical analysis of glucose translocation using both the native Cryo-EM structure (7WSN) and the AI-predicted AlphaFold model using Steered Molecular Dynamics (SMD) at physiological (310.15 K) and attenuated (303.15 K) temperatures with two different allosteric modulators, ATP and ADP. While the AlphaFold model managed to capture baseline static topologies, dynamic profiling revealed that it intrinsically over-optimizes for static stability, immediately collapsing into a sterically occluded, hyper-packed artifact. Translocation through this constricted geometry forces severe steric solvent exclusion, abruptly stripping the substrate’s hydration shell and resulting in immediate and immense thermodynamic friction (>300 kJ/mol). Under ATP-bound physiological strain, this hyper-bonded structural clamp prevents functional relaxation, ultimately inducing a catastrophic kinematic failure. Furthermore, ensemble Dynamic Cross-Correlation Matrix (DCCM) analysis demonstrates that the AI-predicted model suffers from persistent rigid-body locking, forcing the transmembrane domain to behave as a kinetically trapped conformation. In contrast, the functionally hydrated Cryo-EM architecture actively maintains a dual-gating pore and successfully isolates the exact mechanical lever driving transport: a native Proline hinge (PRO379) that seamlessly routes allosteric signals from the nucleotide anchor to the pore gate. Within the AlphaFold model, this critical allosteric wiring is disrupted, forcing mechanical coupling through an unphysiological, hyper-correlated aromatic pathway. These findings yield novel atomistic insights into GLUT4 gating mechanics, while definitively establishing that rigid AI-generated architectures lack the essential internal free volume and conformational plasticity required to sustain accurate thermodynamic profiling of dynamic membrane transporters.
This work introduces BEAM, a multiscale framework that learns slow collective variables from coarse-grained simulations to guide all-atom enhanced sampling to explain how vectorial secretion accelerates pertactin folding by excluding an off-pathway kinetic trap.
Lan Yang, Qing Luan, Michael C. Baxa et al.· bioRxiv· 0 citations
Human aquaporins are integral membrane proteins that facilitate transmembrane transport of small molecules. Among the 13 members of this family, pH-regulated human aquaporin-10 (hAQP10) plays a critical role in glycerol metabolism and lipid homeostasis. A molecular-level understanding of water and glycerol transport through hAQP10 nanopores is essential for enabling rational therapeutic interventions. We present an all-atom molecular dynamics (MD) simulation study characterizing the nanoscale structure, thermodynamic stability, and glycerol permeability of tetrameric hAQP10 channels embedded in lipid bilayer membranes. Using equilibrium and advanced sampling MD simulations, we investigate the cooperative diffusion of water and glycerol through the channel. Free energy (ΔG) landscape derived from replica-exchange umbrella sampling simulations reveals multiple binding sites and energy barriers of a few kBT along the channel axis. Integrating the (ΔG) profile with the inhomogeneous solubility-diffusion model, we estimate a single channel diffusive permeability of glycerol through hAQP10 to be 4 × 10-17 cm3/s at a concentration of 100 mM. Glycerol residence times in the channel during unbiased simulations range from nanoseconds to microseconds. Transport kinetics, characterized using a theoretical model derived by coarse-graining all-atom simulation trajectories, indicate a mean first-passage time of several microseconds. Steered molecular dynamics simulations comparing water and glycerol permeation energetics reveal similar barriers in the open conformation. Together, these findings provide a comprehensive quantitative picture of glycerol and water permeation through hAQP10, with potential implications in understanding physiological role of hAQP10 in human health.
Kunal Rai, Himanshu Joshi· Proceedings of the National...· 0 citations
Cold regulated protein 15A (COR15A) is a late embryogenesis abundant (LEA) protein from Arabidopsis thaliana that is expressed in response to low temperatures and dehydration. Here, we use a combination of ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulations at elevated temperatures for enhanced sampling to explore the conformational landscape of COR15A. The IM-MS data display charge states from +5 to +12, and collision cross section (CCS) distributions indicative of two dominant coexisting conformational families centered on approximately 1200 and 1800 Å2, with subconformers evident. The structures sampled with MD are histogrammed, which provides a pseudo-CCS distribution (pCCS) highly similar to experimental IM-MS data, allowing us to compare with small-angle X-ray scattering-derived structures of COR15A in functional conditions. This approach gives compelling evidence that the experimental gas-phase data correspond to natively accessible states. In contrast to commonly used biophysical approaches, IM-MS permits simultaneous observation of disordered forms as well as conformers populated upon dehydration, allowing insights into dehydration-responsive LEA proteins.
Dale Stuchfield, Ana Bath Alén, Matthew Venables et al.· Journal of Physical Chemistr...· 0 citations
Membrane fusion is an essential process in cells that requires a balance of lipid composition to establish biophysical properties conducive to topology changes. During fusion, lipids of opposing membranes must invert and overcome energy barriers associated with forming highly curved stalk and pore intermediates. While theoretical work has modelled the effect of lipid intrinsic curvature on stalk formation, quantifying the relationship experimentally has proven to be a challenge due to the inability to vary lipid curvature without concomitantly changing other properties that affect fusion. Here we address this hurdle by using hydrostatic pressure to modulate lipid intrinsic curvature independently of chemical composition. Using high-pressure stopped-flow fluorimetry, we measured rates of calcium-mediated lipid mixing between populations of vesicles, a process that is strongly inhibited by pressure. We correlated mean lipid intrinsic curvature across pressure with lipid mixing rates by incorporating complementary small-angle x-ray scattering measurements for each individual lipid component. This analysis showed that lipid mixing rates, a proxy for hemifusion, across compositional and pressure regimes are determined by changes in lipid spontaneous curvature. Consistent with previous theoretical models, we find a linear relation between lipid intrinsic curvature and the hemifusion stalk formation energy, offering direct experimental support for the stalk hypothesis. Significance statement Membrane fusion proceeds through a hemifusion stalk intermediate whose formation energy depends on lipid intrinsic curvature, a central prediction of the stalk hypothesis that has lacked direct experimental support. Previous tests relied on changes in lipid composition that affect multiple membrane properties, confounding the contribution of curvature alone. Here we use hydrostatic pressure to tune lipid curvature independently of chemical composition and calibrate its effects with high-pressure SAXS. Hemifusion rates across three lipid compositions and four pressures collapse into a single exponential dependence on mean spontaneous curvature, yielding a linear relation between curvature and energy consistent with continuum elastic theory. This work quantifies how lipid composition tunes fusion kinetics, suggesting that small changes in lipid curvature may strongly affect fusogenicity.
D. Milshteyn, Jacob R. Winnikoff, J. Morgan et al.· bioRxiv· 0 citations
It is suggested that cold adaptation in pBGL may involve not only enhanced flexibility but also network-mediated organization of conformational dynamics, providing a structural framework for understanding the activity–stability balance of cold-active enzymes.
Xian He, Mengting Liu, Xintong Li et al.· Frontiers in Microbiology· 1 citation
Assembly energetics of transmembrane proteins occurs through the orchestrated molecular interplay of the protein's membrane partitioning free energy and the mechanical and physicochemical properties of the bilayer. While amphipathic residues help accommodate diametrically opposite forces and the steep energy gradient at the membrane-water interface, the magnitude to which the intrinsic curvature and elastic energy of the bilayer regulates protein thermodynamics at the interface remains poorly understood. Here, using PagP as a model β-barrel, we compare the per-residue energetics of an interface site across membranes with(out) distinct curvature stress, lipid packing, and headgroup chemistry. In vesicles, the measured interface energetics correlates well with known free energy scales. Interestingly, unlike vesicles, we show that planar bicelles impose fundamentally distinct membrane-protein coupling regimes: (i) PagP experiences altered folding with a stabilized membrane-associated intermediate and (ii) side chain transfer free energies are restructured in a residue-specific manner. Additionally, the energetics are unaffected by the presence of nonlamellar lipids. Our results reveal an unexpected dependence of partitioning energetics on both bilayer elastic energy and lipid packing defects, demonstrating how subtle changes in membrane physical properties could redirect protein folding pathways and thermodynamics. Our findings underscore the need for caution when extrapolating side chain transfer free energies from simplified model membranes to crowded, compositionally complex in vivo environments.
Swadha Gupta, R. Mahalakshmi· Biochemistry· 0 citations
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