High-field EPR and site-directed spin labeling reveal a periodical polarity profile: The sequence 88 to 94 of the phototransducer NpHtrII in complex with sensory rhodopsin, NpSRII

Abstract

Taking advantage of the improved spectral resolution of high-field electron paramagnetic resonance (EPR) at 95 GHz/3.4 Tas compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the polarity profile in a protein-protein interface is obtained. Nitroxide spin label side chains are introduced at positions 88 to 94 in the AS-1 sequence of the membrane adjacent HAMP domain of the transducer protein, NpHtrII, which is reconstituted in complex with sensory rhodopsin, NpSRII from Natronobacterium pharaonis. Position-dependent variations of the values of the nitroxide magnetic tensor components g.~, and A.: suggest that the spin label side chains at positions 88 to 93 of AS-1 are located between a hydrophobic anda hydrophilic microenvironment. The observed periodicity of the polarity properties of the respective spin label microenvironment agrees with an ~t-helical secondary structure of this part of AS-I and validates a recently published molecular model which locates residues 88 and 91 in the interface between helices F and G of NpSRII and AS-1 of NpHtrII close to the cytoplasmic lipid-water interface.

Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis

“…5B), suggesting the presence of an ␣-helical structure, also demonstrated by high field EPR analysis (34). Starting from position 95, where the two spectral components are clearly distinguishable, the mobility data are biased toward dynamic component 2 (13).…”

“…During the fitting procedure, the g tensor values, the values of A xx and A yy of the hyperfine tensor and the Lorentzian and Gaussian line width parameters were fixed to the values found for the corresponding reference spectra of the singly labeled species. The g tensor parameters used were determined by fitting the low temperature spectra detected at 95 GHz (34). A xx and A yy were fixed to 0.58 and 0.42 mT, respectively, whereas A zz was variable to account for differences in the polarity of the immediate spin label environment (34,35).…”

“…The g tensor parameters used were determined by fitting the low temperature spectra detected at 95 GHz (34). A xx and A yy were fixed to 0.58 and 0.42 mT, respectively, whereas A zz was variable to account for differences in the polarity of the immediate spin label environment (34,35). The spectra were convoluted with a field-independent line shape function composed of a superposition of 29% Lorentzian and 71% Gaussian of 0.3 and 0.37 mT widths, respectively.…”

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

“…The polarity parameter values for the sequence 88 to 94 in the first HAMP domain of NpHtrII in complex with NpSRII are shown in Figure 5A (Brutlach et al, 2006). It is evident that positions 90 and 93 in NpHtrII are located in a more polar, water accessible environment.…”

High-Frequency EPR Instrumentation

“…By balancing the paths lengths associated with the IF source, the video contribution of the phase noise of this DRO (-80 dBc at 10 kHz) could be suppressed by more than 46 dB. Steinhoff et al [100] used the same design in their 95-GHz instrument. Prisner et al [38] used this principle in their 180-GHz pulse EPR spectrometer.…”