2D DARR spectra of U-¹³C-¹⁵N labeled ubiquitin in frozen H₂O solution at 264 K and 213 K.

<p>A) Spectrum recorded at 264 K sample temperature (red). B) Spectrum recorded at 213 K sample temperature (blue). The 1D spectra shown are extracted form the δ₁ slices indicated inside the 2D spectra. The two spectra illustrate the extent of line broadening induced by lowering the temperature in frozen protein solutions.</p

Temperature dependence of ¹³C linewidth of perdeuterated ubiquitin in frozen H₂O solution.

<p>Spectra were recorded on a 750 MHz spectrometer, at 12 kHz MAS, using 1024 acquisitions. The increase in linewidth from 256 K to 202 K is similar to the line broadening observed on fully protonated ubiquitin. These data prove that imperfect decoupling is not the reason for the increase in linewidth.</p

Temperature dependence of ¹³C T₂’ of frozen ubiquitin-water solution in comparison with T₂* calculated from the ¹³C linewidth.

<p>T₂’ shown with black circles were measured with a simple Hahn echo pulse sequence and the data were fit to exponential decay curves. The values T₂* plotted with red diamonds were calculated from the Cδ1 linewidth of Ile23. The T₂’s measured with an echo sequence <i>increase</i> moderately towards lower temperatures and can, therefore, not explain the line broadening observed at lower temperatures.</p

Temperature dependence of ¹³C linewidth in an ubiquitin-water solution.

<p>Spectra were recorded on a 750 MHz spectrometer, at 12 kHz MAS, using 128 acquisitions. Direct ¹³C excitation by a 90° pulse was used at 279 K, a ¹H-¹³C CP pulse sequence was used otherwise. The spectra at 279 K and 264 K were recorded with less sample than the others explaining the reduced signal intensity. The spectra show that the ¹³C lines broaden abruptly when the solution freezes between 279 K and 264 K. After that, the linewidth increases in a continuous manner with lowering the temperature.</p

Temperature dependence of the ¹H and ²H water line intensity in frozen ubiquitin solution.

<p>The ¹H line intensity of the non-frozen water obtained by applying a sine bell square window function followed by magnitude mode processing (blue squares) becomes diminishing small around 240 K. The Intensity of non-frozen water ²H line of frozen ubiquitin-D₂O solution (red diamonds) shows similar temperature dependence. Both ¹H and ²H data were normalized to the respective signal intensity above the freezing point and expressed as g water per g protein. These data show that the non-frozen water becomes hard to detect in frozen ubiquitin solutions at about 240 K when observed with ¹H and ²H NMR.</p

Change in ¹³C line shape of an ubiquitin-water solution with temperature.

<p>Fits of the Ile23 Cδ1 line of ubiquitin in frozen H₂O solution to either Gaussian (blue) or Lorentzian (red) line shapes at two different temperatures. The residuals are shown with dashed lines in the same color. At 258 K the line can be fit better with a Lorentz function (χ² = 5.7*10¹⁴) than with a Gaussian (χ² = 1.9*10¹⁵) indicating a dominantly T₂ relaxation based linewidth. At 213 K the line fits better to a Gauss (χ² = 5.5*10¹⁴) than to a Lorentz (χ² = 1.3*10¹⁵) function indicating that chemical shift distributions are more important for the linewidth at low temperatures. Also note the chemical shift change of ∼0.5 ppm.</p

Functional Model of Metabolite Gating by Human Voltage-Dependent Anion Channel 2

Voltage-dependent anion channels (VDACs) are critical regulators of outer mitochondrial membrane permeability in eukaryotic cells. VDACs have also been postulated to regulate cell death mechanisms. Erastin, a small molecule quinazolinone that is selectively lethal to tumor cells expressing mutant RAS, has previously been reported as a ligand for hVDAC2. While significant efforts have been made to elucidate the structure and function of hVDAC1, structural and functional characterization of hVDAC2 remains lacking. Here, we present an in vitro system that provides a platform for both functional and structural investigation of hVDAC2 and its small molecule modulator, erastin. Using this system, we found that erastin increases permeability of VDAC2 liposomes to NADH in a manner that requires the amino-terminal region of VDAC2. Furthermore, we confirmed that this VDAC2-lipsome sample is folded using solid-state NMR

Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array

The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly ¹³C- and ¹⁵N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR

Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags

Dynamic nuclear polarization is an emerging technique for sensitizing solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable biradicals, the polarization source for the cross effect mechanism, are typically codissolved at millimolar concentrations with proteins of interest. Here we describe the high-affinity biradical tag TMP-T, created by covalently linking trimethoprim, a nanomolar affinity ligand of dihydrofolate reductase (DHFR), to the biradical polarizing agent TOTAPOL. With TMP-T bound to DHFR, large enhancements of the protein spectrum are observed, comparable to when TOTAPOL is codissolved with the protein. In contrast to TOTAPOL, the tight binding TMP-T can be added stoichiometrically at radical concentrations orders of magnitude lower than in previously described preparations. Benefits of the reduced radical concentration include reduced spectral bleaching, reduced chemical perturbation of the sample, and the ability to selectively enhance signals for the protein of interest

NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples

We characterize the effect of specifically bound biradicals on the NMR spectra of dihydrofolate reductase from <i>E. coli</i>. Dynamic nuclear polarization methods enhance the signal-to-noise of solid state NMR experiments by transferring polarization from unpaired electrons of biradicals to nuclei. There has been recent interest in colocalizing the paramagnetic polarizing agents with the analyte of interest through covalent or noncovalent specific interactions. This experimental approach broadens the scope of dynamic nuclear polarization methods by offering the possibility of selective signal enhancements and the potential to work in a broad range of environments. Paramagnetic compounds can have other effects on the NMR spectroscopy of nearby nuclei, including broadening of nuclear resonances due to the proximity of the paramagnetic agent. Understanding the distance dependence of these interactions is important for the success of the technique. Here we explore paramagnetic signal quenching due to a bound biradical, specifically a biradical-derivatized trimethoprim ligand of <i>E. coli</i> dihydrofolate reductase. Biradical-derivatized trimethoprim has nanomolar affinity for its target, and affords strong and selective signal enhancements in dynamic nuclear polarization experiments. In this work, we show that, although the trimethoprim fragment is well ordered, the biradical (TOTAPOL) moiety is disordered when bound to the protein. The distance dependence in bleaching of NMR signal intensity allows us to detect numerous NMR signals in the protein. We present the possibility that static disorder and electron spin diffusion play roles in this observation, among other contributions. The fact that the majority of signals are observed strengthens the case for the use of high affinity or covalent radicals in dynamic nuclear polarization solid state NMR enhancement