17 research outputs found
Combining EPR spectroscopy and X ray crystallography to elucidate the structure and dynamics of conformationally constrained spin labels in T4 lysozyme single crystals
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling is used to investigate the structure and dynamics of conformationally constrained spin labels in T4 lysozyme single crystals. Within a single crystal, the oriented ensemble of spin bearing moieties results in a strong angle dependence of the EPR spectra. A quantitative description of the EPR spectra requires the determination of the unit cell orientation with respect to the sample tube and the orientation of the spin bearing moieties within the crystal lattice. Angle dependent EPR spectra were analyzed by line shape simulations using the stochastic Liouville equation approach developed by Freed and co-workers and an effective Hamiltonian approach. The gain in spectral information obtained from the EPR spectra of single crystalline samples taken at different frequencies, namely the X-band and Q-band, allows us to discriminate between motional models describing the spectra of isotropic solutions similarly well. In addition, it is shown that the angle dependent single crystal spectra allow us to identify two spin label rotamers with very similar side chain dynamics. These results demonstrate the utility of single crystal EPR spectroscopy in combination with spectral line shape simulation techniques to extract valuable dynamic information not readily available from the analysis of isotropic systems. In addition, it will be shown that the loss of electron density in high resolution diffraction experiments at room temperature does not allow us to conclude that there is significant structural disorder in the system
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
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 of 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 being characterized by a bent conformation 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
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