High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin

Abstract

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…A study of phospholamban (PLB) illustrates this point, where global analysis of spectra at two frequencies yielded values for rotational correlation times and order parameters that were much more precisely determined than at either frequency alone [34]. Other studies have also demonstrated that multi-frequency EPR is an appealing approach to circumvent the low resolution at X-band for sub-nanosecond time scales [29,31,36,73].…”

“…High fields provide increased sensitivity to measure the structural parameters of membranes and membraneassociated proteins, such as fluidity, polarity, orientation and membrane order [26,27,30,31,74,75]. An improved sensitivity of sample holders at high fields will greatly facilitate the unraveling of these complex parameters.…”

“…High-field EPR approaches offer the possibility of increased signal sensitivity and information content, thus potentially extending the scope of research aimed at biological macromolecules. High fields increase EPR sensitivity and broaden its practice through improved g-factor resolution for extracting biophysical constraints, which include topics such as dynamics of molecular motion, local polarity and hydrogen bonding, relative orientation information, and membrane ordering [26][27][28][29][30][31][32][33].…”

Site-Directed Spin Labeling and Electron Paramagnetic Resonance (EPR) Spectroscopy: A Versatile Tool to Study Protein-Protein Interactions

“…The A zz component can be obtained from cw X-band EPR spectra of spin labeled proteins in frozen samples. The principal g-tensor components and their variation can be determined with high accuracy using high-field EPR techniques due to the enhanced Zeeman resolution (Steinhoff et al, 2000b). In regular secondary structure elements with anisotropic salvation (e.g., surface exposed α-helices), the water density and hence the tensor component values A zz and g xx are a periodic function of residue number.…”

SDSL-ESR-based protein structure characterization

“…First, two parameters are used to parameterize the partial averaging of the rotational motion within a cone model, i.e., by defining the anisotropy of the cone with the opening angle # 0 and an asymmetry angle u 0 . Second, the traces of the interaction tensors g and A are linearly corrected with the parameters p A (Marsh 1981) and Prot (Steinhoff et al 2000) that take into account the effects of polarity and proticity, respectively. Third, when calculating the convolution of the magnetic field distribution and basic lineshape, two linewidth parameters are also used: a single (effective) rotational correlation time, s c , and an additional broadening constant W. The rotational correlation time defines a Lorentzian-type lineshape in the motional narrowing approximation (Nordio 1976), while the additional broadening constant arises primarily from unresolved hydrogen superhyperfine interactions and contributions from paramagnetic impurities (e.g., oxygen), external magnetic field inhomogeneities, field modulation effects, and spin-spin interaction.…”