Research ArticleAPPLIED SCIENCES AND ENGINEERING

Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix

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Science Advances  04 Oct 2019:
Vol. 5, no. 10, eaay1394
DOI: 10.1126/sciadv.aay1394
  • Fig. 1 The self-resonant microhelix.

    (A) A fabricated five-turn microhelix wrapped around a 0.4-mm–outer diameter capillary. During fabrication, the microhelix is tightly wound around a 0.4-mm drill bit and glued inside a Rexolite cylinder. The drill bit is removed, and the glue is allowed to dry for several days. The microhelix assembly is placed in (B) a coupling and support assembly, which includes a planar microcoupler. (C) The planar microcoupler consists of a stripline impedance match to an inductive coupling loop. SMA, SubMiniature version A. (D) Finite-element modeling simulations of the microwave magnetic field, normalized to input power, at 9.5 GHz show an active region of good magnetic field homogeneity over a 0.8-mm height. The measured microwave magnetic field of 3.2 G/W1/2 corresponds to a 20-ns π/2 pulse at approximately 20 mW. Dimensions of the microhelix, where the self-resonance is determined by the capacitance formed between each turn and the inductance of the windings, are shown. The frequency can be tuned during fabrication by the number of turns, the pitch of the turns, or the inner diameter. The microwave characteristics of the fabricated microhelix can be found in table S1.

  • Fig. 2 The molecular structure of the [FeFe]-hydrogenase active site, the H-cluster.

    Highlighted are the proximal and distal irons, Fep and Fed, respectively, the cyanide ligand (CNd), and the ADT ligand. S, yellow; Fe, orange; N, blue; C, tan; O, red. Structure is from Protein Data Bank (PDB) ID 4XDC.

  • Fig. 3 Frozen solution EPR on an 85-nl-volume sample at X-band.

    Three EPR experiments performed with a 0.4 mm inner diameter self-resonant microhelix. Shown are the (A) continuous-wave (CW), (B) real (Re.) and imaginary (Im.) nonadiabatic rapid scan (NARS), and (C) field-swept two-pulse ESE EPR experiments of the tyrosine D radical (YD) in photosystem II with 85 nl of frozen solution sample at a temperature of 80 K. Calculated MDIFF pseudo-modulation of 0.5 mT is shown for the NARS and field-swept ESE experiments to directly compare to the continuous-wave EPR experiment. The total time for the experiments was 49, 55, and 45 min, respectively. The signal-to-noise ratio is calculated and tabulated in Table 2.

  • Fig. 4 Single-crystal continuous-wave EPR of YD in the photosystem II core complex.

    Continuous-wave EPR collected with the 0.4 mm inner diameter self-resonant microhelix at two angles of the photosystem II YD radical from a single crystal at a temperature of 80 K. The crystal dimensions were 0.3 mm by 0.18 mm by 0.18 mm. Shown in red is a fitted simulation with similar features. A nonspecifically bound Mn2+ signal is also present in the mother liquor of the crystal, indicated by an asterisk (∗). Each spectrum was collected in 49 min with a signal-to-noise ratio of approximately 35.

  • Fig. 5 Pulse EPR on a single crystal of the H-cluster in [FeFe]-hydrogenase.

    (A) The molecular structure of the [FeFe]-hydrogenase active site, the H-cluster, from PDB ID 4XDC is shown with the molecular frame located with the distal iron (Fed) as the origin. The molecular frame rotational coordinates can be found in table S2. S, yellow; Fe, orange; N, blue; C, tan; O, red. (B) The P1211 symmetry schematic relating the molecular frame (x, y, z) to the crystal frame (a, b, c) and, last, to the laboratory system frame (L1, L2, L3) is shown. The two molecular frames from the asymmetric unit are present in Site I and can be translated to Site II by crystal symmetry operations. (C) The static magnetic field (B0) is positioned along the L1 axis, while the microwave magnetic field (B1) can be either along the L2 axis or along the L3 axis. A rotation of 180° is feasible around the L3 axis, but only a partial rotation around the L2 axis is feasible because of the B1 rotating with the crystal resulting in B1 to become parallel to B0. A third partial rotation is feasible if the sample is rotated by 90° around the L2 axis. (D) Pulse EPR experiments collected with the 0.4 mm inner diameter self-resonant microhelix with a [FeFe]-hydrogenase single crystal of C. pasteurianum (CpI) in the Hox state showing collected data in one plane for a full rotation of 180° in 5° steps at a temperature of 15 K. The crystal dimensions were approximately 0.3 mm by 0.1 mm by 0.1 mm, and each spectrum was collected in 8 min with a signal-to-noise ratio of approximately 290. (E) A stereo view of the analyzed g-tensor (gx, red; gy, green; and gz, blue) is mapped on the crystal structure (PDB ID: 4XDC). For a three-dimensional (3D) view of the proposed g-tensor, see https://act-epr.org/FeFeHydrogenase.html.

  • Fig. 6 Single-crystal HYSCORE EPR of the H-cluster in [FeFe]-hydrogenase.

    Top left: Field-swept two-pulse ESE EPR spectrum at 150°. The figure labels (A, B, and C) are representative of the spectral peaks. The HYSCORE spectra collected with the 0.4 mm inner diameter self-resonant microhelix of a [FeFe]-hydrogenase single crystal of C. pasteurianum (CpI) in the Hox state at an orientation of 150° collected at a temperature of 15 K. The 2D density representation shows correlations between the nuclear spin transitions in both projections of the electronic spin. (A) Clean HYSCORE spectrum due to the peak corresponding to only one of the EPR signals in the unit cell of the crystal. The correlated features between these transitions are indicated by the white, red, and green circles. (B) Relatively featureless HYSCORE spectrum suggests little hyperfine interaction at this orientation. (C) HYSCORE on two overlapping EPR signals representing different orientations of the enzyme molecule with respect to the magnetic field. The HYSCORE was set up using the Bruker HYSCORE wizard with the following settings: π/2, 40 ns; τ, 280 ns; and Δτ, 48 ns with 256 points each and 20 shots per point. Each HYSCORE spectrum was collected in approximately 1 hour.

  • Table 1 Resonator EPR signal characteristics calculated and measured using a power saturation measurement of a lithium phthalocyanine point sample.

    Dimensions of the resonators and further experimental details are provided in the Supplementary Materials.

    GeometryUnsat.
    signal
    Sat.
    signal
    Eff.
    η
    Q0-value
    Meas.
    ηQ0
    Calc.Meas.Calc.Meas.
    Bruker MD5W11.01.01.01.06.12 × 10−666501.0
    Bruker MS31.51.21.01.079.6 × 10−66001.17
    PMR RO6010LM4.41.20.91.24.63 × 10−3616.94
    PMR sapphire18.613.33.93.84.63 × 10−318120.59
    Microhelix35.728.26.15.76.63 × 10−322035.84
  • Table 2 Signal-to-noise calculations for the three experiments performed on the photosystem II YD radical in frozen solution at a temperature of 80 K.

    Approximately 1.6 × 1012 spins were calculated to be in the 85 nl that fill the microhelix. SNR, signal-to-noise ratio.

    ExperimentSNR Re.SNR Im.Time
    Continuous wave19713149 min
    NARS4400230055 min
    NARS (MDIFF)410423
    ESE24845 min
    ESE (MDIFF)106

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaay1394/DC1

    EPR characteristics comparison of multiple resonators

    Power saturation and Λave measurements

    Sensitivity comparison of the X-band microhelix to a commercial dielectric resonator

    Qualitative sensitivity comparison of the X-band microhelix to high-frequency single-mode resonators

    Crystal rotation and simulation

    Fig. S1. Dimensions and geometry of the four resonators compared in this paper.

    Fig. S2. Ansys HFSS finite-element modeling simulation of the microwave magnetic fields comparing the PMR and microhelix.

    Fig. S3. Power saturation curve of LiPC using various resonators.

    Fig. S4. Continuous-wave EPR of frozen solution photosystem II BBY particles performed in the Bruker MD5W1 dielectric resonator at a temperature of 80 K.

    Fig. S5. Comparison of the g-tensor proposed by Adamska-Venkatesh et al. (38) and the current proposed g-tensor from this work.

    Table S1. Resonator characteristics calculated and measured.

    Table S2. Rotational matrices for the crystal frame with respect to the laboratory frame and the g-tensor with respect to the molecular frame.

    References (4456)

  • Supplementary Materials

    This PDF file includes:

    • EPR characteristics comparison of multiple resonators
    • Power saturation and Λave measurements
    • Sensitivity comparison of the X-band microhelix to a commercial dielectric resonator
    • Qualitative sensitivity comparison of the X-band microhelix to high-frequency single-mode resonators
    • Crystal rotation and simulation
    • Fig. S1. Dimensions and geometry of the four resonators compared in this paper.
    • Fig. S2. Ansys HFSS finite-element modeling simulation of the microwave magnetic fields comparing the PMR and microhelix.
    • Fig. S3. Power saturation curve of LiPC using various resonators.
    • Fig. S4. Continuous-wave EPR of frozen solution photosystem II BBY particles performed in the Bruker MD5W1 dielectric resonator at a temperature of 80 K.
    • Fig. S5. Comparison of the g-tensor proposed by Adamska-Venkatesh et al. (38) and the current proposed g-tensor from this work.
    • Table S1. Resonator characteristics calculated and measured.
    • Table S2. Rotational matrices for the crystal frame with respect to the laboratory frame and the g-tensor with respect to the molecular frame.
    • References (4456)

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