MOMD Analysis of NMR Line Shapes from Aβ-Amyloid Fibrils: A New Tool for Characterizing Molecular Environments in Protein Aggregates

The microscopic-order-macroscopic-disorder (MOMD) approach for ²H NMR line shape analysis is applied to dry and hydrated 3-fold- and 2-fold-symmetric amyloid-Aβ₄₀ fibrils and protofibrils of the D23N mutant. The methyl moieties of L17, L34, V36 (C–CD₃), and M35 (S–CD₃) serve as probes. Experimental ²H spectra acquired previously in the 147–310 K range are used. MOMD describes local probe motion as axial diffusion (<i><b>R</b></i> tensor) in the presence of a potential, <i>u</i>, which represents the spatial restrictions exerted by the molecular surroundings. We find that <i>R</i>_∥ = (0.2–3.3) × 10⁴ s^–1, <i>R</i>_⊥ = (2.2–2.5) × 10² s^–1, and <b><i>R</i></b> is tilted from the ²H quadrupolar tensor at 60–75°. The strength of <i>u</i> is in the (2.0–2.4) <i>kT</i> range; its rhombicity is substantial. The only methyl moieties affected by fibril hydration are those of M35, located at fibril interfaces. The associated local potentials change form abruptly around 260 K, where massive water freezing occurs. An independent study revealed unfrozen “tightly-peptide-bound” water residing at the interfaces of the 3-fold-symmetric Aβ₄₀ fibrils and at the interfaces of the E22G and E22Δ Aβ₄₀-mutant fibrils. Considering this to be the case in general for Aβ₄₀-related fibrils, the following emerges. The impact of water freezing is transmitted selectively to the fibril structure through interactions with tightly-peptide-bound water, in this case of M35 methyl moieties. The proof that such waters reside at the interfaces of the 2-fold-symmetric fibril, and the protofibril of the D23N mutant, is new. MOMD provides information on the surroundings of the NMR probe directly via the potential, <i>u</i>, which is inherent to the model; a prior interpretation of the same experimental data does so partially and indirectly (see below). Thus, MOMD analysis of NMR line shapes as applied to amyloid fibrils/protein aggregates emerges as a consistent new tool for elucidating the properties of, and processes associated with, molecular environments in the fibril

Local Ordering at Mobile Sites in Proteins from Nuclear Magnetic Resonance Relaxation: The Role of Site Symmetry

Restricted motions in proteins (e.g., N–H bond dynamics) are studied effectively with NMR. By analogy with restricted motions in liquid crystals (LC), the local ordering has in the past been primarily represented by potentials comprising the <i>L</i> = 2, |<i>K|</i> = 0, 2 spherical harmonics. However, probes dissolved in LCs experience nonpolar ordering, often referred to as <i>alignment</i>, while protein-anchored probes experience polar ordering, often referred to as <i>orientation</i>. In this study we investigate the role of local (site) symmetry in the context of the polarity of the local ordering. We find that potentials comprising the <i>L</i> = 1, |<i>K|</i> = 0, 1 spherical harmonics represent adequately polar ordering. It is useful to characterize potential symmetry in terms of the irreducible representations of <i>D</i>_2<i>h</i> point group, which is already implicit in the definition of the rotational diffusion tensor. Thus, the relevant rhombic <i>L</i> = 1 potentials have <i>B</i>_1<i>u</i> and <i>B</i>_3<i>u</i> symmetry whereas the relevant rhombic <i>L</i> = 2 potentials have <i>A</i>_<i>g</i> symmetry. A comprehensive scheme where local potentials and corresponding probability density functions (PDFs) are represented in Cartesian and spherical coordinates clarifies how they are affected by polar and nonpolar ordering. The Cartesian coordinates are chosen so that the principal axis of polar axial PDF is pointing along the <i>z</i>-axis, whereas the principal axis of the nonpolar axial PDF is pointing along ±<i>z</i>. Two-term axial potentials with 1 ≤ <i>L</i> ≤ 3 exhibit substantial diversity; they are expected to be useful in NMR-relaxation-data-fitting. It is shown how potential coefficients are reflected in the experimental order parameters. The comprehensive scheme representing local potentials and PDFs is exemplified for the <i>L</i> = 2 case using experimental data from ¹⁵N-labeled plexin-B1 and thioredoxin, ²H-, and ¹³C-labeled benzenehexa-<i>n</i>-alkanoates, and nitroxide-labeled T4 lysozyme. Future prospects for improved ordering analysis based on combined atomistic and mesoscopic approaches are delineated

A New Wavelet Denoising Method for Experimental Time-Domain Signals: Pulsed Dipolar Electron Spin Resonance

We adapt a new wavelet-transform-based method of denoising experimental signals to pulse-dipolar electron-spin resonance spectroscopy (PDS). We show that signal averaging times of the time-domain signals can be reduced by as much as 2 orders of magnitude, while retaining the fidelity of the underlying signals, in comparison with noiseless reference signals. We have achieved excellent signal recovery when the initial noisy signal has an SNR ≳ 3. This approach is robust and is expected to be applicable to other time-domain spectroscopies. In PDS, these time-domain signals representing the dipolar interaction between two electron spin labels are converted into their distance distribution functions <i>P</i>(<i>r</i>), usually by regularization methods such as Tikhonov regularization. The significant improvements achieved by using denoised signals for this regularization are described. We show that they yield <i>P</i>(<i>r</i>)’s with more accurate detail and yield clearer separations of respective distances, which is especially important when the <i>P</i>(<i>r</i>)’s are complex. Also, longer distance <i>P</i>(<i>r</i>)’s, requiring longer dipolar evolution times, become accessible after denoising. In comparison to standard wavelet denoising approaches, it is clearly shown that the new method (WavPDS) is superior

Improved Sensitivity for Long-Distance Measurements in Biomolecules: Five-Pulse Double Electron–Electron Resonance

We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron–electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both

Pulsed Dipolar Spectroscopy Reveals That Tyrosyl Radicals Are Generated in Both Monomers of the Cyclooxygenase‑2 Dimer

Cyclooxygenases (COXs) are heme-containing sequence homodimers that utilize tyrosyl radical-based catalysis to oxygenate substrates. Tyrosyl radicals are formed from a single turnover of substrate in the peroxidase active site generating an oxy-ferryl porphyrin cation radical intermediate that subsequently gives rise to a Tyr-385 radical in the cyclooxygenase active site and a Tyr-504 radical nearby. We have utilized double-quantum coherence (DQC) spectroscopy to determine the distance distributions between Tyr-385 and Tyr-504 radicals in COX-2. The distances obtained with DQC confirm that Tyr-385 and Tyr-504 radicals were generated in each monomer and accurately match the distances measured in COX-2 crystal structures

Pulsed ESR Dipolar Spectroscopy for Distance Measurements in Immobilized Spin Labeled Proteins in Liquid Solution

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron–electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized

Synthesis and Solution-Phase Characterization of Sulfonated Oligothioetheramides

Nature has long demonstrated the importance of chemical sequence to induce structure and tune physical interactions. Investigating macromolecular structure and dynamics is paramount to understand macromolecular binding and target recognition. To that end, we have synthesized and characterized flexible sulfonated oligothioetheramides (oligoTEAs) by variable temperature pulse field gradient (PFG) NMR, double electron–electron resonance (DEER), and molecular dynamics (MD) simulations to capture their room temperature structure and dynamics in water. We have examined the contributions of synthetic length (2–12mer), pendant group charge, and backbone hydrophobicity. We observe significant entropic collapse, driven in part by backbone hydrophobicity. Analysis of individual monomer contributions revealed larger changes due to the backbone compared to pendant groups. We also observe screening of intramolecular electrostatic repulsions. Finally, we comment on the combination of DEER and PFG NMR measurements via Stokes–Einstein–Sutherland diffusion theory. Overall, this sensitive characterization holds promise to enable de novo development of macromolecular structure and sequence–structure–function relationships with flexible, but biologically functional macromolecules

HAMP Domain Conformers That Propagate Opposite Signals in Bacterial Chemoreceptors

<div><p>HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.</p> </div

Inter-spin distance measurements by PDS.

<p>Shown are experimentally determined distances of spin-labeled proteins and Cα–Cα distances from the Aer2 1–172 crystal structure. The values shown in parentheses refer to the width (Å) at half the maximum peak height, and qualify peak broadening and conformational heterogeneity. Small values represent narrow peaks and a homogeneous conformation. Large values represent broad peaks consistent with more heterogeneous populations.</p>a<p>Attachment of the MTSSL spin labels can add up to 13 Å to the Cα–Cα separation, or equivalently 6.5 Å each.</p