SUMMARY SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured by itself but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remain elusive. Here, we report an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulations systematically characterize the coil-to-molten globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster resonance energy transfer and double electron-electron resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in submillisecond timescales and potentially play a vital role in forming the SNARE complex. We expect that this integrative approach may help further our understanding of IDPs.
Interfacial electrostatic properties of monodisperse silica nanoparticles (SiNPs) in aqueous suspensions as a function of bulk pH were characterized by spin labeling EPR of two ionizable nitroxides: (1) IMTSL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-yl)methylmethanesulfonothioate) and IKMTSL (S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl). SiNPs of ca. 116 nm in diameter (by particle number) were synthesized using the Stober method, and their surface was modified by silanization under harsh conditions to ensure robust attachment of the thiol-terminated ligands to the silica surface. These ligands were consequently modified with either IMTSL or IKMTSL to characterize the surface electrostatic potential of the nanoparticles from their EPR spectra. EPR titration data for these two pH-sensitive nitroxides allowed for differentiating the dielectric and electrostatic contributions to the interfacial properties of SiNPs. From such a titration at room temperature an effective local dielectric constant experienced by IMTSL at the silica nanoparticle−water interface was found to be ε eff = 70.8 ± 5.0 whereas ε eff ≈ 57 ± 4 was found for IKMTSL. Surface electrostatic potential calculated from EPR titration of IKMTSL demonstrated an approximately linear increase in the magnitude starting at about zero at pH ∼4.0 and reaching ∼−150 mV at pH ∼8.5. This is in agreement with the existing literature on the surface potential associated with the silanol deprotonation developing over a wide pH range. While the attachment linker employed for the two nitroxides has some flexibility, it still ensures the location of the pH-sensitive tags close to the surface. For these reasons the values of the electrostatic surface potential reported by these nitroxides are significantly higher than those reported by the zeta potential measurements. Overall, spin labeling methods developed here expand the applicability of spin-labeling EPR to measurements of interfacial electrostatic properties of metal oxide nanoparticles.
FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions -electrical dipoles in case of FRET and magnetic dipoles in case of DEER -between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Molecular vol-
Two new ammonium vanadate hydrates, i.e., M 3 (H 2 O) 2 V 8 O 24 •2NH 4 (M = Mn and Co, I and II, respectively) were synthesized using hydrothermal reaction conditions, and their structures were determined by single crystal X-ray diffraction [I: P2/ m (No. 10), Z = 1, a = 8.2011(2) Å, b = 3.5207(1) Å, c = 9.9129(3) Å, β = 110.987(2)°; II: C2/m (No. 12), Z = 2, a = 19.4594(6) Å, b = 6.7554(2) Å, c = 8.4747(3) Å, β = 112.098(2)°]. Interestingly, the two structures are homeotypic, with the structure of I exhibiting an uncommon type of structural disorder between locally-bridging Mn(H 2 O) 2 2+ (i.e., part of the oxide framework) and nonbridging NH 4+ cations over the same site (1:2 ratio), wherein two NH 4 + ions occupy the same site as the two H 2 O molecules when Mn(II) is vacant. The amount of Mn(II) in the formula of I was determined by a combination of techniques, including electron paramagnetic resonance, while the relative amounts of NH 4 + /H 2 O in its structure were determined by combined thermogravimetric-mass spectrometry analyses as well as confirmed by infrared spectroscopy. In contrast, this site disorder is absent in the crystal structure of II, which contains a fully ordered arrangement of locally-bridging Co(H 2 O) 2 2+ and NH 4 + cations that alternate down its c-axis within a larger superstructure related to I by (a → c, b → 2b, c → 2a). Within both structures, the respective Mn 2+ /Co 2+ cations bridge to neighboring edge-sharing chains of distorted VO 5 square pyramids, forming a three-dimensional network that contains channels of H 2 O and NH 4 + molecules. Hydrogen bonding distances in I are significantly longer and weaker than in II and leading to the disordered structure of I. Both show the loss of all H 2 O and NH 4 + molecules, by ∼300 °C for I and a slightly higher ∼325 °C for II, in each case yielding V 2 O 5 and MV 2 O 6 (M = Co or Ni) as the final products. Both I and II exhibit visiblelight bandgap sizes of ∼1.55 and ∼1.77 eV, respectively, owing to low-energy metal-to-metal electronic transitions. Further, I shows a temperature-dependent photocatalytic activity (at 40 °C) for the production of hydrogen from the reduction of water under irradiation by UV−vis or only visible light at respective rates of ∼314 μmol H 2 g −1 h −1 and ∼54 μmol H 2 g −1 h −1 (irradiant power density of ∼1.0 W/cm 2 ). Thus, these first two known ammonium vanadate hydrates provide new insights into the structural driving forces for ordered versus disordered structures as well as into their resulting physical properties.
Due to their ubiquity, versatility, and chemical diversity, lipid bilayers are an increasingly important component of engineered biosensors and biophysical measurement platforms. Bilayers supported on solid substrates are a common design feature of these systems, and the presence of the substrate introduces perturbations in the bilayer structure, affecting both stability and function.
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