Changes in the Microenvironment of Nitroxide Radicals around the Glass Transition Temperature

Abstract

For structural characterization by pulsed EPR methods, spin-labeled macromolecules are routinely studied at cryogenic temperatures. The equilibration of the conformational ensemble during shock-freezing occurs to a good approximation at the glass transition temperature (Tg). In this work, we used X-band power saturation continuous wave (cw) EPR to obtain information on the glass transition temperatures in the microenvironment of nitroxide radicals in solvents or bound to different sites in proteins. The temperature dependence of the saturation curve of nitroxide probes in pure glycerol or ortho-terphenyl showed detectable transitions at the respective Tg values, with the latter solvent characterized by a sharper change of the saturation properties, according to its higher fragility. In contrast, nitroxide probes in a glycerol/water mixture showed a discontinuity in the saturation properties close to the expected glass transition temperature, which made the determination of Tg complicated. Low-temperature W-band cw EPR and W-band ELDOR-detected NMR experiments demonstrated that the discontinuity is due to local rearrangements of H-bonds between water molecules and the nitroxide reporter group. The change in the network of H-bonds formed between the nitroxide and water molecules that occurs around Tg was found to be site-dependent in spin-labeled proteins. This effect can therefore be modulated by neighboring residues with different steric hindrances and/or charge distributions and possibly by the glycerol enrichment on protein surfaces. In conclusion, if the thermal history of the sample is carefully reproduced, the nitroxide probe is extremely sensitive in reporting site-specific changes in the H-bonding to water molecules close to Tg and local glass transition temperatures in spin-labeled macromolecules.

Biomolecular EPR Meets NMR at High Magnetic Fields

“…The resolution of the EDNMR experiment also allows the different spectral contributions to be resolved. In the case of SDSL labeled proteins, discrete populations of nitroxides exposed to the EDNMR spectra recorded in the high magnetic field region are of particular importance, as they allow the precise values of nitrogen hyperfine (A zz ), and quadrupole, (P zz ), tensor z-components to be read out [236,558,562,563], see Figure 9a. These parameters are most sensitive to probe the microenvironment of nitroxide radicals.…”

“…The high resolution of EDNMR also allows characterizing the changes of the nitroxide microenvironment upon the changes of the sample conditions. From a comparison of ENDMR spectra recorded in shock-frozen samples, which were subsequently annealed at higher temperatures, it was shown that the H-bond network, in which the nitroxide is involved, rearranges around the glass transition temperature [563]. The spectra were recorded close to the gzz, MI = −1 spectral position at 50 K. The experimental spectra (black traces) are overlaid with best-fit Gaussian lines that are assigned to nitroxide radicals forming either zero (red trace), one (green trace) or two (blue trace) H-bonds with the solvent molecule.…”

“…The fits were performed on the first derivative mode of the experimental traces. For details see [562,563].…”

“…At this field range, the Larmor frequency, ν n , of many nuclei is significantly larger than 10 MHz, allowing for their detection by EDNMR. Over the last decade, a series of reports appeared that used high-field EDNMR to characterize a number of chemical systems and materials with a wide variety of nuclei in radical [228,236,545,[557][558][559][560][561][562][563][564] and transition metal containing systems [272,273,275,276,[565][566][567][568][569][570][571][572][573][574][575][576][577][578][579][580][581][582][583][584].…”

“…We also note that at high field, correlation transitions (those involving multiple nuclei) can be more easily suppressed that can contribute to the observed linewidth at lower microwave bands (X-, Q-band). In the text below we describe recent results from our laboratories on using EDNMR to characterize radicals and paramagnetic metal complexes at higher magnetic fields [236,273,274,545,558,559,[562][563][564]572,583]. For further information about the EDNMR method we refer the reader to recent reviews [274,585].…”