Internal dynamics of the 3-Pyrroline-N-Oxide ring in spin-labeled proteins

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy

tRNA^Sec constructs for crystallization screening.

<p>RNA 1 represents full-length tRNA^Sec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for <i>in vitro</i> transcription, which allowed synthesis of tRNA^Sec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).</p

Hydration and metal ion binding of mouse tRNA^Sec.

<p>(<b>A</b>) Hydration of the G27•U43 wobble bps in molecules A (carbon – gold) and B (carbon – silver) of mouse tRNA^Sec, showing a full complement of first-shell water molecules (cyan spheres). Hydrogen bonds are indicated by dashed lines. (<b>B</b>) Anomalous difference Fourier map contoured at the 5 σ level (green mesh) calculated with anomalous differences recorded from a Mn²⁺-soaked crystal and phases obtained from molecular replacement with the native structure as a search model. Molecule A – gold; molecule B – silver. There are three common Mn²⁺ sites (1–3) in the two tRNA^Sec molecules. Sites 4 and 5 were found only in chain B. The boxed region is shown in a close-up view in the following panel. (<b>C</b>) Close-up of the boxed region of panel (B). Mn²⁺ ion (site 4; purple sphere) apparently reinforcing the interaction of U9 (AD-linker) with A48•C45 (first bp of the variable arm).</p

Anticodon loop conformations in mouse tRNA^Sec.

<p>Stereo stick model of the closed anticodon loop conformation of molecule A (<b>A</b>) and of the open anticodon loop conformation of molecule B (<b>B</b>). The 2′-oxygen of the U34 ribose in molecule A that is methylated in a subset of cellular tRNA^Sec is shown as a thicker stick. Water molecules – cyan spheres. Hydrogen bonds are indicated by dashed lines.</p

Non-denaturing RNA purification.

<p>(<b>A</b>) Elution profile of <i>in vitro</i> transcribed mouse tRNA^Sec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (<b>B</b>) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (<b>C</b>) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (<b>D</b>) Elution profile of mouse tRNA^Sec from a Superdex 75 10/300 GL column.</p

Analysis of purified RNA.

<p>(<b>A</b>) Native and (<b>B</b>) denaturing PAGE analysis of fractions collected from Superdex 75 10/300 GL gel filtration. S – concentrated RNA sample after anion exchange chromatography. (<b>C</b>) Analysis of mouse tRNA^Sec by multi-angle static light scattering. The optical density at 260 nm (OD₂₆₀; magenta), Rayleigh ratio (Rq; blue) and differential refractive index (RI; yellow) were monitored during analytical gel filtration on a Superdex 200 10/300 GL column. The measurement was done by Wyatt Technology Europe GmbH.</p

Tertiary interactions of the AD-linker and variable arm.

<p>(<b>A</b>) Water-mediated interaction of the AD-linker with the first bp of the variable arm in mouse tRNA^Sec (molecule A – gold; molecule B – silver). Water molecules – cyan spheres. Hydrogen bonds are shown as dashed lines. (<b>B</b>) Close-up of the variable arm (molecule A). Nucleotides of the variable loop are labeled. (<b>C</b>) Superposition of the mouse tRNA^Sec variable loop (carbon – gold) with a stable GAGA tetraloop from the 23S rRNA sarcin/ricin domain (carbon – cyan; PDB ID 483D).</p

Crystal contacts of ^ΔGCCAtRNA^Sec.

<p>Stacking interactions of the terminal G1-C72 bp of the acceptor stem of molecule A with nucleotides G19 and U20 of the the D-loop of a neighboring molecule A. Left – overview. Right – close-up of the crystal packing interaction. In this and the following figures, atoms are color-coded in identical fashion; carbon – gold (or as the respective molecule); nitrogen – blue; oxygen – red; phosphorus – orange. Hydrogen bonds are indicated by dashed lines. The right view is rotated 45° about the horizontal axis as indicated.</p

Internal Dynamics of the 3‑Pyrroline‑<i>N</i>‑Oxide Ring in Spin-Labeled Proteins

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy