Detection of correlated conformational fluctuations in intrinsically disordered proteins through paramagnetic relaxation interference.

Functionally relevant conformational states of intrinsically disordered proteins (IDPs) are typically concealed in a vast space of fast interconverting structures. Here we present a novel methodology, NMR-based paramagnetic relaxation interference (PRI), that allows for direct observation of concerted motions and cooperatively folded sub-states in IDPs. The proposed NMR technique is based on the exploitation of cross correlated electron-nuclear dipolar relaxation interferences in doubly spin-labeled proteins and probes the transient spatial encounter of electron-nucleus spin pairs.

Evidence for disorder in L-alanine lattice detected by Pulsed-EPR spectroscopy at cryogenic temperatures.

The unusual behavior of lattice dynamics of L-alanine has been assigned to intermolecular dynamics and localization of vibrational energy. Recent heat capacity and Pulsed-EPR measurements support presence of thermally activated dynamic orientational disorder in the L-alanine lattice below 20 K. In the present study, the additional evidence for possible thermally activated disordered behavior of L-alanine lattice have been obtained by investigating dependences of longitudinal relaxation time of first stable L-alanine radical, SAR1, on sample cooling rates for the same low temperature interval. The obtained relaxation time by Pulsed-EPR shows clear dependence on cooling rates and this behavior can be explained within two types of suggested spin-lattice relaxation mechanisms for the paramagnetic centers in the hydrogen-bonded organic crystal.

Thermal stability of radiation-induced free radicals in gamma-irradiated L-alanine single crystals.

Decay of the radiation-induced stable free radicals in l-alanine single crystals and powders at the temperatures from 379 to 476 K was examined by electron paramagnetic resonance. For single crystals, the calculated activation energy of the radical decay is 104.3+/-1.7 kJ/mol (i.e. 12 538+/-202 K) and the frequency factor lnv (O) is 24.1+/-0.4 min(-1). The lifetime of the radical in single crystals at 296 K is 162 years. The results confirm the long-term stability of the radicals, but the decay was found to be faster in large crystals than in powders.

Heat capacity and electron spin echo evidence for low frequency vibrational modes and lattice disorder in L-alanine at cryogenic temperatures.

With the view of understanding the low frequency (40-50 cm(-1)) motional processes in L-alanine around 4 K, we have carried out heat capacity (CP) and electron spin echo (ESE) measurements on L-alanine and L-alanine-d7. The obtained CP data show the so-called boson peak (seen as a maximum in CP/T3 versus T plots) in the low temperature region (1.8-20 K). The phase memory time, T(M), and spin lattice relaxation time, T1, of the spin probe, the so-called first stable alanine radical (SAR1), *CHCH3COOH, have been measured between 4 and 105 K. The obtained relaxation rate 1/T1 shows an anomalous increase which coincides with the emergence of a boson peak in the low temperature region (4-20 K). Together, the ESE and the CP data confirm the existence of a thermally activated dynamic orientational disorder in the lattices of both compounds below 20 K. The results help explain the discrepancy between the CP data from powders and single crystals of alanine, as well as the proanomalous relaxation mechanisms for SAR1 in these lattices, and they also provide a mechanism for the spin-lattice relaxation process for SAR1 at cryogenic temperatures.

A pulse EPR study of longitudinal relaxation of the stable radical in gamma-irradiated L-alanine.

The longitudinal relaxation rate of the first stable alanine radical, SAR1, was studied by employing pulse EPR technique over a wide temperature interval (5-290 K). The complex nonexponential recovery of the longitudinal magnetization in this temperature interval has been described with two characteristic relaxation times, 1/T*(1a) as the faster component and 1/T*(1b) as the slower component, respectively. It was shown that 1/T*(1a) is strongly affected by the CH(3) group dynamics of the SAR1 center. The complete temperature dependence of 1/T*(1a) was described by invoking several relaxation mechanisms that involve hindered motion of the CH(3) group from classical rotational motion to coherent rotational tunneling. It was shown that all relevant relaxation mechanisms are determined by a single correlation time with the potential barrier (Delta E/k=1570 K). On the other hand the temperature dependence of 1/T*(1b) is related to the motional dynamics of the neighborly NH(3) and CH(3) groups. We found a larger average potential barrier for this motion (Delta E/k=2150 K) corresponding to smaller tunneling frequencies of the neighbor groups.

Study of relaxation rates of stable paramagnetic centers in gamma-irradiated alanine.

The stable L-alanine radical induced by gamma-irradiation was examined by electron paramagnetic resonance (EPR), transfer saturation EPR and electron nuclear double resonance (ENDOR) in the temperature region of fast motion of the methyl group (180-320 K). From the obtained spectral line broadening and spectral intensity the correlation time for the methyl rotation was estimated. The complex processes determining the relaxation rate were examined in the same temperature interval. It was shown that important contributions to the relaxation rate arise from non-secular and pseudo-secular types of contributions. The non-secular contribution involves intramolecular dynamics while the pseudo-secular contribution originates from intermolecular motions. The obtained values for the dynamical parameters have been compared with those obtained by pulse EPR methods and by proton nuclear magnetic resonance (NMR) on undamaged crystals.