Slow Dynamics around a Protein and Its Coupling to Solvent

Solvent is essential for protein dynamics and function, but its role in regulating the dynamics remains debated. Here, we employ saturation transfer electron spin resonance (ST-ESR) to explore the issue and characterize the dynamics on a longer (from μs to s) time scale than has been extensively studied. We first demonstrate the reliability of ST-ESR by showing that the dynamical changeovers revealed in the spectra agree to liquid–liquid transition (LLT) in the state diagram of the glycerol/water system. Then, we utilize ST-ESR with four different probes to systematically map out the variation in local (site-specific) dynamics around a protein surface at subfreezing temperatures (180–240 K) in 10 mol % glycerol/water mixtures. At highly exposed sites, protein and solvent dynamics are coupled, whereas they deviate from each other when temperature is greater than LLT temperature (∼190 K) of the solvent. At less exposed sites, protein however exhibits a dynamic, which is distinct from the bulk solvent, throughout the temperature range studied. Dominant dynamic components are thus revealed, showing that (from low to high temperatures) the overall structural fluctuation, rotamer dynamics, and internal side-chain dynamics, in turn, dominate the temperature dependence of spin-label motions. The structural fluctuation component is relatively slow, collective, and independent of protein structural segments, which is thus inferred to a fundamental dynamic component intrinsic to protein. This study corroborates that bulk solvent plasticizes protein and facilitates rather than slaves protein dynamics

Effects of Anisotropic Nanoconfinement on Rotational Dynamics of Biomolecules: An Electron Spin Resonance Study

The development of nanostructured materials for next-generation nanodevice technologies requires a better understanding of dynamics of the objects as confined in nanospace. Here, we characterize the rotational dynamics of a long (14-residue) proline-based peptide (approximately 4 nm in length) under anisotropic nanoconfinement using spin-labeling CW/pulsed ESR techniques as well as spectral simulations. We show by pulsed ESR experiments that the conformations of the peptide in several different nanochannels and a bulk solvent are retained. Parameters characterizing the dynamics of the peptide regarding temperature (200–300 K) and nanoconfinement are determined from nonlinear least-squares fits of theoretical calculations to the multifrequency experimental spectra. Remarkably, we find that this long helical peptide undergoes a large degree of rotational anisotropy and orientational ordering inside the nanochannels, but not in the bulk solvent. The rotational anisotropy of the helical peptide barely changes with the nanoconfinement effects and remains substantial, as the nanochannel diameter is varied from 6.1 to 7.1 and 7.6 nm. This finding is advantageous for addressing purposes of anisotropic nanoconfinement and for advancing our understanding of the rotational dynamics of nano-objects as confined deeply inside the nanostructures of materials

Concurrent Observation of Bulk and Protein Hydration Water by Spin-Label ESR under Nanoconfinement

Under nanoconfinement the formation of crystalline ice is suppressed, allowing the study of water dynamics at subfreezing temperatures. Here we report a temperature-dependent investigation (170–260 K) of the behavior of hydration water under nanoconfinement by ESR techniques. A 26-mer-long peptide and the Bax protein are studied. This study provides site-specific information about the different local hydrations concurrently present in the protein/peptide solution, enabling a decent comparison of the hydration moleculesthose that are buried inside, in contact with, and detached from the protein surface. Such a comparison is not possible without employing ESR under nanoconfinement. Though the confined bulk and surface hydrations behave differently, they both possess a transition similar to the reported fragile-to-strong crossover transition around 220 K. On the contrary, this transition is absent for the hydration near the buried sites of the protein. The activation energy determined under nanoconfinement is found to be lower in surface hydration than in bulk hydration. The protein structural flexibility, derived from the interspin distance distributions <i>P</i>(<i>r</i>) at different temperatures, is obtained by dipolar ESR spectroscopy. The <i>P</i>(<i>r</i>) result demonstrates that the structural flexibility is strongly correlated with the transition in the surface water, corroborating the origin of the protein dynamical transition at subfreezing temperatures

Determination of the n3β-d structure.

<p>(a) The time-domain DEER data for the n3β-d (0.5 mM) in the studied conditions, including the vitrified bulk solvent (sol(s)/H₂O) and the nanochannels (SBA15a and SBA15b). The gray lines represent the exponential baselines that best fit the DEER data. There are two insets. One displays a ribbon model for the n3β-d showing the spin-label side-chains at the 3rd and 9th sites of the peptide. The model was derived from a NMR study (PDB code: 1G04). The other inset shows the baseline-corrected DEER traces for the sol(s)/H₂O and the SBA15a, and also the simulated DEER traces (in green color) using the obtained P(r)s. There are some distinct differences in the two traces. (b) The (normalized) interspin distance distributions of the n3β-d peptides in the conditions studied. The average distances of the three measurements are approximately the same, indicating the n3β structure remains roughly unchanged. A much-broadened P(r) for the bulk solution study is obtained due to the solvent heterogeneity. The inset shows the Pake doublets converted from the DEER data. (c) Cw-ESR spectra of the n3β-d at 50 K. The clustering, caused by the solvent heterogeneity at 50 K, is evidently observed in the cw-ESR spectra of the bulk solution study, but not in the nanochannel studies.</p

Water accessibility study of the PPm3-s by ESEEM.

<p>(a) Three-pulse ESEEM time-domain data (solid lines) after the removal of the exponential decaying function in the raw data. The modulation depth is directly correlated to the peak intensity of the FT-ESEEM and can be quantitatively characterized by the best-fit parameter <i>k_D</i> (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>). The dashed lines represent the theoretical fits to the experimental data using the equation described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. (b) The FT-ESEEM data for the PPm3-s in various deuterated conditions. The peaks correspond to the Larmor frequency of nucleus ²H, indicating the PPm3-s is surrounded by D₂O. The inset shows a ribbon model of a PPm3 variant carrying three spin labels.</p

Side-Chain Packing Interactions Stabilize an Intermediate of BAX Protein against Chemical and Thermal Denaturation

Bcl-2-associated X (BAX) protein plays a gatekeeper role in transmitting apoptotic signaling from cytosol to mitochondria. However, little is known about its stability. This study reports a comprehensive investigation on the stability of BAX using spin-label ESR, CD, and ThermoFluor methods. Point mutations covering all of the nine helices of BAX were prepared. ESR study shows that BAX can be divided into two structural regions, each responding differently to the presence of guanidine hydrochloride (GdnHCl). The N-terminal region (helices 1–3) is denatured in 6 M GdnHCl, whereas the C-terminal region (helices 4–9) is resistant to the denaturing effects. The far-UV CD spectra show an appreciable amount of helical content of BAX at high temperatures. The magnitude of the near-UV CD signal is increased with increasing temperature in either 0 or 6 M GdnHCl, indicating an enhancement of aromatic side-chain packing in the C-terminal region. Taken together with ThermoFluor results, we show that a core interior, wherein aromatic interactions are highly involved, within the C-terminal region plays an important role in stabilizing BAX against the denaturing effects. Collectively, we report a highly stable, indestructible intermediate state of BAX. Side-chain packing interactions are shown to be the major stabilizing force in determining BAX structure

Long-range water accessibility study by ESE.

<p>The theoretical fits (red lines) to the ESE experimental data (blue lines) using a stretched exponential function (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>) as previously described. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Huang1" target="_blank">[11]</a> The results for the n3β-s and PPm3-s are shown in (a) and (b), respectively. The decay signals acquired by the ESE experiments were fitted over the maxima of the deuterium modulation as described in Zecevic et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Zecevic1" target="_blank">[32]</a> to minimize the influence from destructive interference of nuclear modulations. The obtained values of the T_M (in ns) and stretching exponent x are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>. The T_M values can be directly used to yield the surrounding proton density (<i>C_ex</i>; cf. Eq. 2) within the range of ∼2 nm from a nitroxide spin.</p

Parameters obtained in the analyses of the ESE and ESEEM data.^§

§<p>Estimated errors: 5%(T_M), 10%(x), 13% (<i>C_ex</i>), 5% (<i>k_D</i>), 10% (Π). Abbreviations: <b>n3-s-a</b> (the n3-s is within SBA15a containing pure water); <b>n3-s-b</b> (the n3-s is within SBA15b containing pure water); <b>n3-s-sol(s)</b> (the n3-s is in a vitrified bulk solvent containing 40 wt% sucrose, (s), in D₂O or H₂O); <b>PPm3-s-sol(g)</b> (PPm3-s is in a vitrified bulk solvent containing 40v/v% glycerol in H₂O; deuterated glycerol is used if the solvent is D₂O, a condition of which is represented by sol(dg)/D₂O in main text); <b>PPm3-s-sol(s)</b> (PPm3-s is in a vitrified bulk solvent containing 40 wt% sucrose in D₂O or H₂O). In all of the experiments, the surface group of the nanochannels is modified to –SiOD in advance if D₂O is used. See Method for details.</p>#<p>The values of T_M and x are obtained in the analysis of the pulsed ESE measurements using a stretched exponential function, , where τ is the time between the two pulses, x the exponent, and Y(0) is the echo intensity at τ  = 0. The obtained values are used to yield <i>C_ex</i> using Eq. (2). The <i>C_ex</i> represents ESE-based water accessibility within the range of ∼2 nm from the nitroxide spin.</p>¶<p>The <i>k_D</i> values are obtained in the theoretical analysis of the ESEEM measurements as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. The best-fit values for the damping constant (<i>τ₀</i>) and phase (φ) are very close together (2.9∼3.0). The Π represents ESEEM-based water accessibility within the range of ∼0.35 nm from the nitroxide spin.</p

Determination of the n3α-d structure.

<p>(a) The time-domain DEER signals of the studied conditions. The gray lines represent the exponential baselines that best fit the data. Inset shows a ribbon model of the n3α-d derived from NMR data (PDB code: 1M25). (b) The P(r) distributions extracted from the time-domain DEER data by the Tikhonov regularization analysis. The average distances (∼2.0 nm) are consistent with the expectation. (c) The cw-ESR spectra of the n3α-d. The spectra of the bulk solution studies are characterized by a broader linewidth and the spectral heterogeneity (indicated by arrows) as compared to the spectra of the nanochannel studies.</p

Cs₃UGe₇O₁₈: A Pentavalent Uranium Germanate Containing Four- and Six-Coordinate Germanium

A pentavalent uranium germanate, Cs₃UGe₇O₁₈, was synthesized under high-temperature, high-pressure hydrothermal conditions at 585 °C and 160 MPa and structurally characterized by single-crystal X-ray diffraction and infrared spectroscopy. The valence state of uranium was confirmed by X-ray photoelectron spectroscopy and electron paramagnetic resonance. The room-temperature EPR spectrum can be simulated with two components using an axial model that are consistent with two distinct sites of uranium­(V). In the structure of the title compound, each ^[6]GeO₆ octahedron is bonded to six three-membered single-ring ^[4]Ge₃O₉^6– units to form germanate triple layers in the <i>ab</i> plane. Each layer contains nine-ring windows; however, these windows are blocked by adjacent layers. The triple layers are further connected by UO₆ octahedra to form a three-dimensional framework with intersecting six-ring channels along the ⟨11̅0⟩ directions. The Cs⁺ cation sites are fully occupied, ordered, and located in the cavities of the framework. Pentavalent uranium germanates or silicates are very rare, and only two uranium silicates and one germanate analogue have been published. However, all of them are iso-structural with those of the Nb or Ta analogues. In contrast, the title compound adopts a new structural type and contains both four- and six-coordinate germanium. Crystal data of Cs₃UGe₇O₁₈: trigonal, <i>P</i>3̅<i>c</i>1 (No. 165), <i>a</i> = 12.5582(4) Å, <i>c</i> = 19.7870(6) Å, <i>V</i> = 2702.50(15) ų, <i>Z</i> = 6, <i>D</i>_calc = 5.283 g·cm^–3, μ­(Mo Kα) = 26.528 mm^–1, <i>R</i>₁ = 0.0204, <i>wR</i>₂ = 0.0519 for 1958 reflections with <i>I</i> > 2σ­(<i>I</i>). GooF = 1.040, ρ_max,min = 1.018, and −1.823 e·Å^–3