An extensive set of ESR spectra was obtained over a wide range of frequencies (9, 95, 170 and 240 GHz) and temperatures (2 to 32°C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model: the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature, and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2, are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered. KeywordsMTSSL; SRLS; side-chain; conformers; tumbling; Ficoll Correspondence to: Jack H. Freed. SUPPORTING INFORMATION AVAILABLE: They include the fitting procedure for determining magnetic tensor components; details on the global nonlinear least squares (NLLS) fitting; best fit parameters in aqueous and in Ficoll solutions; the two vs. three component fits to spectra of 131R1 and single-component fits to spectra of R2; variation of the reduced χ 2 with the diffusion tilt angle; further discussion of spectra recorded in 65% sucrose solution; contribution of the global tumbling in 25% Ficoll solution. This material is available free of charge via the Internet at

We describe a far-infrared electron-paramagnetic-resonance ͑EPR͒ spectrometer for broadband ͑100-300 GHz͒ use. The spectrometer is operated in the reflection mode and uses broadband quasioptical methods to separate the transmitted from the reflected radiation. We describe and illustrate its operation at 170 GHz ͑1.8 mm͒ and compare its performance to that of our transmission mode spectrometer operating at 250 GHz. We also discuss the advantages of the reflection bridge for performing EPR experiments over a broad range of frequencies, and we consider methods of improving the performance of the bridge. This includes a novel design for variable coupling of the reflection-mode Fabry-Pérot resonator.

The ESR parameters of PC spin labels in frozen membranes do not simply represent the membrane polarity or water penetration profile. Instead, they show a distribution between hydrogen-bonded (HB) and non-hydrogen bonded (non-HB) states, which is affected by a number of factors in the membrane composition. Similar to the exclusion of solutes from crystallizing solvents, the pure bulk gel phase excludes nitroxides, forcing acyl chains to take bent conformations. In these conformations the nitroxide is hydrogen-bonded. Furthermore, upon gradual cooling in the supercooled gel PC labels undergo slow lateral aggregation resulting in a broad background signal. However, if the sample is instantly frozen, this background is replaced by the HB component. In membranes with cholesterol the observed HB/ non-HB ratio can best be described by a partition-like equilibrium between nitroxides located in defects of lipid structure within the hydrophobic core and those close to the membrane surface.