Assessment of Nanosecond Time Scale Motions in Native and Non-Native States of Ubiquitin

作者：Morozova Olga B; Yurkovskaya Alexandra V^*

来源：Journal of Physical Chemistry B, 2015, 119(39): 12644-12652.

DOI：10.1021/acs.jpcb.5b07333

The paramagnetic relaxation times of the aromatic and beta protons of Tyr59 and His68 residues of the native ubiquitin and of Tyr59 residue of the non-native ubiquitin were determined from an analysis of chemically induced dynamic nuclear polarization (CIDNP) kinetics obtained during the photoreaction of the protein and 2,2'-dipyridyl excited in the triplet state. Using the paramagnetic relaxation times determined earlier for the radicals of free amino acids as an internal standard and assuming that the hyperfine interaction (HFI) anisotropy is very similar for the radicals of free amino acids and the corresponding radicals of amino acid residues in the proteins, we determined parameters that characterize the intramolecular mobility of different protons in native and two non-native states of ubiquitin. The latter are denatured at pH 2 and 57 degrees C, and the A-state at pH 2 in a 60%/40% methanol/water mixture. The determination of the two parameters of intramolecular mobility (i.e., the correlation time of internal motion, tau(e), and the order parameter, S-2) was only possible by analyzing paramagnetic relaxation data obtained at two magnetic fields (4.7 and 9.4 T) using nuclear magnetic resonance (NMR) spectrometry. Intramolecular correlation times fall into the submicrosecond microsecond time scale. Longer correlation times and higher order parameters were found for the less accessible Tyr59 residue than for the His68 residue, as well as for the more buried beta protons than for the aromatic protons for both of the protein residues in the native state. For Tyr59, intramolecular mobility increases following the loss of the tertiary structure of ubiquitin. These findings strongly support the reliability of the obtained data.

• 出版日期2015-10-1