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Pulsed dipolar spectroscopy, such as double electron–electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate the effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function. [ABSTRACT FROM AUTHOR]

While activation of BAX is required for initiating mitochondria-mediated apoptosis, the underlying mechanisms remain unsettled. We studied conformations of BAX protein using pressure- and temperature-resolved ESR techniques and obtained the thermodynamic properties of the conformations. We show that inactive BAX is structurally heterogeneous and exists in equilibrium between two major populations of the conformations, UM and UM′, of which the former is thermodynamically favored at room temperature. An increase in the population of UM′, induced by either pressure or point mutations of BAX, renders BAX susceptible to oligomerization, which leads to cell death. This study uncovers the biological significance of BAX conformations and shows that the pro-apoptotic activity of BAX can be triggered by altering the equilibrium between the two states. It suggests that therapeutic intervention may focus on shifting the balance in the conformational heterogeneity. [ABSTRACT FROM AUTHOR]

Upon apoptotic stress, Bcl-2 associated X (BAX) protein undergoes conformational changes and oligomerizes, leading to the mitochondrial membrane permeabilization and cell death. While structures of the resultant oligomer have been extensively studied, little is known about the intermediates that describe the reaction pathway from the inactive monomers to activated oligomers. Here we characterize the intermediate structures of BAX using combined small-angle X-ray scattering (SAXS) with on-line gel-filtration and electron spin resonance (ESR). The intermediates, including monomers, dimers, and tetramers, are reconstructed via integrating the SAXS-envelopes and ESR-determined skeleton structures. The hence revealed structures suggest a linear oligomerization of BAX utilizing the extended dimers with the two flexible α6 chains protruded out as ditopic ligands. The results of molecular dynamics simulation also support the ditopic dimer conformation with mobile α6. The ditopic dimers could further wind into a helical rod structure with three dimers in one helical turn. Our results not only reveal the on-pathway intermediates, but also suggest a ditopic oligomerization mechanism that may bridge the observed intermediate structures in solution to the large BAX assemblies lately observed on mitochondria. [ABSTRACT FROM AUTHOR]

We have synthesized and investigated a series of yellow-emitting Sr3Ce(PO4)3:Eu2+ phosphors, showing an extreme broadband at around 535 nm (with a FWHM of ca. 200 nm/6760 cm−1) and a large Stoke shift (ca. 8265 cm−1), which is attributed to the 4f–5d transition of Eu2+ without the contribution from Ce3+ emission under excitation at long wavelengths (＞370 nm). However, such broadband Eu2+ emission is rather surprising, considering that there is only one cation site for the Eu2+ luminescent center. Herein, we investigated the crystal structure by performing X-ray Rietveld refinement on the synchrotron X-ray diffraction data, and demonstrate that both cation and anion sites in Sr3Ce(PO4)3 are disordered in the host lattice. These unusual structural characters generate a variety of distinct Eu2+ sites, which is verified by decay lifetime analysis and electron paramagnetic resonance spectra, and thus result in astonishing broadband yellow-emission. Moreover, a white LED device with a color-rendering index of 86.5, a color temperature of 5996 K, and chromaticity coordinates of (0.32, 0.38) was obtained by combining a 405 nm near-UV LED chip and the phosphor blends of yellow-emitting Sr3Ce(PO4)3:Eu2+ and the commercial blue-emitting BaMgA10O17:Eu2+ phosphor. These results indicate that the as-prepared yellow-emitting Sr3Ce(PO4)3:Eu2+ phosphor has potential applications in the dual-color-phosphor-converted WLEDs. [ABSTRACT FROM AUTHOR]

Nanochannels of mesoporous silica materials were previously found useful for reducing the tumbling motion of encapsulated biomolecules while leaving the biomolecular structure undisturbed. Here we show that experiments of cw-ESR distance measurement in nano-confinement can benefit immediately from the above mentioned features of sufficiently slow molecular tumbling, enabling more accurate determination of interspin distances throughout the temperature range, from 200 to 300 K. A 26-residue prion protein peptide, which can fold into either a helical or hairpin structure, as well as its variants, are studied by using ESR. By comparing the spectra obtained in vitrified bulk solutions vs.mesopores, the spectra from the latter display typical slow-motional lineshapes, thereby enabling dipolar anisotropy to be unambiguously revealed throughout the temperature range, whereas the spectra from the former are dominated by the disordering of the side chain and the rotational tumbling of the peptide. The spectral changes regarding the two secondary structures in nano-confinement are found to show a strong correlation with the dynamic properties of the backbones. The effect of viscosity agent perturbation on the motion of an R1 nitroxide side chain, a commonly employed probe, could be substantial in a bulk solution condition, though it is absolutely absent in nanochannels. Under nano-confinement, the probe is proven sufficiently sensitive to the backbone motions. Overall, the distance distributions determined from the mesopore studies not only describe the conformational structures (by average distances), but also the backbone dynamics (by distribution widths) of the spin-labeled peptides. [ABSTRACT FROM AUTHOR]

Identifying protein dynamics is essential for studying protein function. However, the time-scale of dynamic modes varies over domains and segments of a protein. Here we describe an approach using multifrequency ESR with mesoporous materials for protein dynamics in confined nanospace that may mimic the crowded nature within a cell where proteins evolve to fold. While multifrequency ESR permits the separation of dynamic motions in different time-scales, we demonstrate its capability to capture dynamics can still be significantly enhanced by the encapsulation of nitroxide-labeled macromolecule into mesopores. Two mutants of a 26-residue prion protein peptide at temperatures from 2 to 27 °C are studied. The nanochannel provides the peptide with an ordered environment such that the global tumbling of peptide is slow, and ‘frozen’ on the ESR timescale. The local dynamic modes of the peptide in nanochannel are, therefore, distinctly reported on the spectra. The spectra of the peptide in β-hairpin vs.α-helical forms differ markedly, demonstrating the significant improvement of ESR spectroscopic capability due to our methodology. Such distinctly different spectral patterns between the two secondary structures of the peptide cannot be obtained from ESR studies in viscous aqueous solution. The dynamic modes on the peptide are thus unambiguously identified in our multifrequency experiments at the X- and Q-bands. Additionally, the multifrequency spectra for each mutant and temperature are simultaneously fitted to the rigorous models, e.g.the slowly-relaxing-local-structure model, for slow-motion ESR. Marked correlations are revealed and characterized quantitatively for the backbone flexibility between the β-hairpin and α-helical forms of the prion protein peptide. Confirmation of the slow collective dynamic modes extending across the β-hairpin is also provided through the spectral simulations. [ABSTRACT FROM AUTHOR]

Multivalent ligands hold promise for enhancing avidity and selectivity to simultaneously target multimeric proteins, as well as potentially modulating receptor signaling in pharmaceutical applications. Essential for these manipulations are nanosized scaffolds that precisely control ligand display patterns, which can be achieved by using polyproline oligo-helix macrocyclic nanoscaffolds via selective binding to protein oligomers and cell surface receptors. This work focuses on synthesis and structural characterization of different-sized polyproline tri-helix macrocyclic (PP3M) scaffolds. Through combined analysis of circular dichroism (CD), small- and wide-angle X-ray scattering (SWAXS), electron spin resonance (ESR) spectroscopy, and molecular modeling, a non-coplanar tri-helix loop structure with partially crossover helix ends is elucidated. This structural model aligns well with scanning tunneling microscopy (STM) imaging. The present work enhances the precision of nanoscale organic synthesis, offering prospects for controlled ligand positioning on scaffolds. This advancement paves the way for further applications in nanomedicine through selective protein interaction, manipulation of cell surface receptor functions, and developments of more complex polyproline-based nanostructures., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)