Alexey Potapov

Nanometer-scale distance measurements in proteins using Gd3+ spin labeling

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings.

High-Field EPR Reveals the Strongly Temperature-Dependent Exchange Interaction in “Breathing” Crystals Cu(hfac)2LR

In the overwhelming majority of the exchange-coupled clusters investigated in field of molecular magnetism, the exchange interaction is constant on temperature. “Breathing” crystals of composition Cu(hfac)2LR undergo temperature-induced reversible structural rearrangements accompanied by significant changes of the effective magnetic moment. Using high-field (W-band) EPR, we provide a solid proof of drastic temperature dependence of exchange interaction J(T) in these compounds that originates from temperature dependence of inter-spin distances. Strong dependence J(T) revealed by EPR makes Cu(hfac)2LR breathing crystals interesting and promising systems in the research toward creation of molecular-magnetic switches and related spin devices.

Successive Stages of Amyloid-β Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization.

Self-assembly of amyloid-β (Aβ) peptides in human brain tissue leads to neurodegeneration in Alzheimers disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Aβ self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Aβ (Aβ40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel β-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.

Solid state nuclear magnetic resonance with magic-angle spinning and dynamic nuclear polarization below 25 K

We describe an apparatus for solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS) at 20-25 K and 9.4 Tesla. The MAS NMR probe uses helium to cool the sample space and nitrogen gas for MAS drive and bearings, as described earlier, but also includes a corrugated waveguide for transmission of microwaves from below the probe to the sample. With a 30 mW circularly polarized microwave source at 264 GHz, MAS at 6.8 kHz, and 21 K sample temperature, greater than 25-fold enhancements of cross-polarized (13)C NMR signals are observed in spectra of frozen glycerol/water solutions containing the triradical dopant DOTOPA-TEMPO when microwaves are applied. As demonstrations, we present DNP-enhanced one-dimensional and two-dimensional (13)C MAS NMR spectra of frozen solutions of uniformly (13)C-labeled l-alanine and melittin, a 26-residue helical peptide that we have synthesized with four uniformly (13)C-labeled amino acids.

Temperature-Dependent Exchange Interaction in Molecular Magnets Cu(hfac)2LR Studied by EPR: Methodology and Interpretations

Exchange-coupled spin triads nitroxide-copper(II)-nitroxide are the key building blocks of molecular magnets Cu(hfac)(2)L(R). These compounds exhibit thermally induced structural rearrangements and spin transitions, where the exchange interaction between spins of copper(II) ion and nitroxide radicals changes typically by 1 order of magnitude. We have shown previously that electron paramagnetic resonance (EPR) spectroscopy is sensitive to the observed magnetic anomalies and provides information on both inter- and intracluster exchange interactions. The value of intracluster exchange interaction is temperature-dependent (J(T)), that can be accessed by monitoring the effective g-factor of the spin triad as a function of temperature (g(eff)(T)). This paper describes approaches for studying the g(eff)(T) and J(T) dependences and establishes correlations between them. The experimentally obtained g(eff)(T) dependences are interpreted using three different models for the mechanism of structural rearrangements on the molecular level leading to different meanings of the J(T) function. The contributions from these mechanisms and their manifestations in X-ray, magnetic susceptibility and EPR data are discussed.

Dynamic nuclear polarization-enhanced 1H–13C double resonance NMR in static samples below 20 K

We demonstrate the feasibility of one-dimensional and two-dimensional ¹H-¹³C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both ¹H-¹³C cross-polarization and ¹H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for ¹H-¹³C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T₂ relaxation of ¹³C, induced by electron spin flips. Carr-Purcell experiments and numerical simulations of Carr-Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on ¹³C-labeled biomolecules was demonstrated with a two-dimensional ¹³C-¹³C exchange spectrum of selectively ¹³C-labeled β-amyloid fibrils.

Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids.

We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D (13)C-(13)C exchange spectroscopy to probe the peptide backbone torsion angles (φ, ψ) in a series of selectively (13)C-labeled 40-residue β-amyloid (Aβ(1-40)) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39 T and 8 K, using a static double-resonance NMR probe and low-power microwave irradiation at 264 GHz. In frozen solutions of Aβ(1-40) fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16-21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl (13)C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous (13)C lineshapes are incorporated in the simulations. The experimental 2D (13)C-(13)C exchange spectra place constraints on the φ and ψ angles between the two carbonyl labels. Although the data are not sufficient to determine φ and ψ uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D (13)C-(13)C exchange spectra can be obtained from a 3.5 mg sample of Aβ(1-40) fibrils in 4 h or less, despite the broad (13)C chemical shift anisotropy line shapes that are observed in static samples.

A triple resonance hyperfine sublevel correlation experiment for assignment of electron-nuclear double resonance lines.

A new, triple resonance, pulse electron paramagnetic resonance (EPR) sequence is described. It provides spin links between forbidden electron spin transitions (DeltaM(S)=+/-1, DeltaM(I) not equal 0) and allowed nuclear spin transitions (DeltaM(I) = +/-1), thus, facilitating the assignment of nuclear frequencies to their respective electron spin manifolds and paramagnetic centers. It also yields the relative signs of the hyperfine couplings of the different nuclei. The technique is based on the combination of electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected NMR experiments in a way similar to the TRIPLE experiment. The feasibility and the information content of the method are demonstrated first on a single crystal of Cu-doped L-histidine and then on a frozen solution of a Cu-histidine complex.

Quantitative characterization of the Mn2+ complexes of ADP and ATPγS by W-band ENDOR

W-band (95 GHz) pulsed electron nuclear double resonance (ENDOR) measurements were carried out to determine quantitatively the first coordination shell of Mn2+ with ADP and ATPγS. The intensity of the ENDOR effect was used for counting the number of equivalent phosphate oxygens and water ligands. Titration curves for determining the binding constant of Mn2+. ADP were obtained using the intensity of the X-band EPR spectrum and the31P ENDOR effect. Both curves gave the same binding constant showing that phosphate ligand counting is plausible, provided that an appropriate reference is available. The comparison of the31P ENDOR effect of the 1:1 ADP and ATPγS complexes shows that two phosphates are coordinated in both; while in ADP they are equivalent, in ATPγS they are slightly different. The reference system for water ligand counting was Mn(H2O)62+ in a H2O-D2O mixture. The results show a smaller error for the2H ENDOR effect, compared to the1H ENDOR effect. Unlike the31P ENDOR effect, the1H ENDOR effect dependence on [ADP] in the titration experiments showed that it is sensitive to variations in the zero-field splitting, which in turn alters the contributions of transitions other than the ‖−1/2>↔‖1/2>. This results in a larger error in the determination of the number of water ligands.