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

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Crystal structure of p115^GHR.

<p>(A) Stereo view of the overall structure. The protein is composed of 11 armadillo repeats. (B) Structure-based sequence alignment of the p115 armadillo repeats and the loop regions. Consensus residues that define the conserved hydrophobic residues of each armadillo repeat are highlighted in blue; amino acids that define conserved polar, neutral residues are highlighted in green. Glycine and proline residues are highlighted in brown and olive, respectively. The repeat numbers are shown on the left. The sequences that form helices H1, H2, and H3 are indicated as green, blue and yellow cylinders. The corresponding ARM loops are marked on top, the USO helix is indicated as red cylinder. (C) N-terminal armadillo-like helical domain (left) and C-terminal Uso1 head domain (right) of p115^GHR, elongated loops with at least 5 residues are colored in red (H1 green, H2 blue, H3 yellow).</p

Dimeric arrangement of the p115 globular head region.

<p>(A) <i>B</i>-factor representation (“S-“ and “W-view”) of p115^GHR molecules aligned by crystal symmetry. The intermolecular contact area (blue) is among the most rigid parts of the structure. (B) Depending on the orientation, the crystallographic dimer of p115^GHR has a single-head (“O-view”) or double-lobed globular appearance (“W-” and ”V-view”).</p

Interaction of p115^GHR and the COG complex subunit COG2.

<p>(A) Electrostatic surfaces of p115^GHR. The amino terminus of the molecule is at the top of the figure. Blue indicates positive charge and red negative charge at the level of 10 kT/e. (B) Conserved residues of p115 and the yeast homolog Uso1p form a highly charged surface of exposed helices which define the COG2 binding site.</p

Model of the overall fold of the full-length p115.

<p>Surface and cartoon representations of a model of the full-length general vesicular transport factor p115 generated by manually fitting a coiled-coil of appropriate length to the C-termini of p115^GHR in the crystallographic dimer. The different views of the p115 model closely resemble published electron micrographs of p115 and Uso1p.</p

Electrostatic surfaces comparison of the superhelical grooves of the p115^GHR, β-catenin and karyopherin-α.

<p>Blue indicates positive charge and red negative charge at the level of 10 kT/e. The amino terminus of the molecule is at the top of the figure.</p

Schematic overview of full-length p115.

<p>The construct comprising p115^GHR used for crystallization is shown in gray.</p

Tracking Transient Conformational States of T4 Lysozyme at Room Temperature Combining X‑ray Crystallography and Site-Directed Spin Labeling

Proteins are dynamic molecules that can transiently adopt different conformational states. As the function of the system often depends critically on its conformational state a rigorous understanding of the correlation between structure, energetics and dynamics of the different accessible states is crucial. The biophysical characterization of such processes is, however, challenging as the excited states are often only marginally populated. We show that a combination of X-ray crystallography performed at 100 K as well as at room temperature and EPR spectroscopy on a spin-labeled single crystal allows to correlate the structures of the ground state and a thermally excited state with their thermodynamics using the variant 118R1 of T4 lysozyme as an example. In addition, it is shown that the surrounding solvent can significantly alter the energetic as well as the entropic contribution to the Gibbs free energy without major impact on the structure of both states

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)-0

<p><b>Copyright information:</b></p><p>Taken from "X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)"</p><p>BMC Structural Biology 2005;5():21-21.</p><p>Published online 14 Dec 2005</p><p>PMCID:PMC1352370.</p><p>Copyright © 2005 Manjasetty et al; licensee BioMed Central Ltd.</p>MLCK (PDB 1FHG). The β-strands are labeled according to Ig fold I set nomenclature. The N-terminal 14 residues and the adhesion recognition RGD motif are highlighted. : Ribbon diagram of the ΔAPEG-1 monomer. The front sheet (strands A'GFCC') and back sheet (strands ABED), are colored purple and pink, respectively. The 3helix is shown in orange