Control of Solvent Dynamics around the B₁₂-Dependent Ethanolamine Ammonia-Lyase Enzyme in Frozen Aqueous Solution by Using Dimethyl Sulfoxide Modulation of Mesodomain Volume

Abstract

The temperature-dependent structure and dynamics of two concentric solvent phases, the proteinassociated domain (PAD) and mesodomain, that surround the protein, ethanolamine ammonialyase (EAL) from Salmonella typhimurium in frozen polycrystalline aqueous solution are addressed by using electron paramagnetic resonance (EPR) spectroscopy of the paramagnetic nitroxide spin probe, TEMPOL, over the temperature (T) range, 195-265 K. Dimethyl sulfoxide (DMSO; added at 0.5, 2.0 and 4.0 % v/v), and present at the maximum freeze concentration at T≤245 K, varies the volume of the interstitial aqueous-DMSO mesodomain (V meso), relative to a fixed PAD volume (V PAD). The increase in V meso /V PAD from 0.8 to 6.0 is quantified by the partitioning of TEMPOL between the two phases. As V meso /V PAD is increased, Arrhenius parameters for activated TEMPOL rotational motion in the mesodomain remain uniform, while the parameters for TEMPOL in the PAD show a progressive transformation toward the mesodomain values (higher-mobility). An order-disorder transition (ODT) in the PAD is detected by exclusion of TEMPOL from the PAD into the mesodomain. The ODT T value is systematically lowered by increased V meso /V PAD (from 215 to 200 K), and PAD ordering kinks the mesodomain Arrhenius dependence. Thus, there is reciprocity in PAD-mesodomain solvent coupling. The results are interpreted as a dominant influence of ice-boundary confinement on PAD solvent structure and dynamics, that is transmitted through the mesodomain, and which decreases with mesodomain volume at increased added DMSO. The systematic tuning of PAD and mesodomain solvent dynamics by variation of added DMSO is an incisive approach for resolution of contributions of protein-solvent dynamical coupling to EAL catalysis.

Confinement dependence of protein-associated solvent dynamics around different classes of proteins, from the EPR spin probe perspective

“…The inter-phase partition coefficient of TEMPOL in Region III is unity. 20 The mean PAD thickness is estimated by using V PAD and the known parameters for the proteins from eqn (2):where ASA is the accessible surface area per protein, 45,46 determined by using Virtual Molecular Dynamics (VMD), 47 and n P is the number of proteins in the 0.3 mL sample volume. The mean t PAD values for the globular proteins, EAL (6.9 Å), SA (5.8 Å), and Mb (3.3 Å) correspond to one to two water molecules (water molecule, van der Waals diameter, 2.8 Å), in agreement with values determined for the protein hydration layer by other methods.…”

“…44 Comparison of the plots of log t c for the proteins with two motional components in Fig. 4(A), (B) and 5(A), (B) shows that they subscribe to the Region I-IV paradigm, 20 to within the 5 K measurement resolution, and that the general trends in the T-dependence of both log t c,s , log t c,f and the compensating W s , W f values are common among the different proteins, under the different confinement conditions. The results in Fig.…”

“…20 EPR sample preparation and mesodomain parameters are collected in Table S2 (ESI †). The protein concentration, DMSO volume (V DMSO ), and TEMPOL concentration in the samples, denoted as ([protein], V DMSO , [TEMPOL]) are as follows: EAL (20 mM, 6.0 mL, 0.2 mM), 20 SA (131 mM, 6.0 mL, 0.2 mM), Mb (150 mM, 1.5.0 mL, 0.05 mM), Cas (24 mM, 0.20 mL, 0.02 mM), PRM (388 mM, 6.0 mL, 0.05 mM), Ab16 (787 mM, 6.0 mL, 0.02 mM). The EPR samples were prepared aerobically, on ice in vials, mixed, and then loaded into quartz EPR tubes (Wilmad-LabGlass, 4 mm outer diameter).…”

“…20 The ice boundary confinement effect on PAD dynamics saturates at added 2% v/v DMSO, which corresponds to an aqueous–DMSO mesodomain shell of mean 20 Å thickness. 20 Thus, in addition to the confinement control and spin probe localization in the protein-vicinal solvent regions, the low- T mesodomain system has the advantages toward study of native features of protein–solvent dynamical coupling of a fully-hydrated protein interior, conditions conducive to formation of a complete hydration layer or PAD, and a surrounding fluid mesodomain with tunable confinement. 21 Further, the linear Arrhenius relation for radical rearrangement kinetics in EAL extends from 295 K through the range of dual-detectable TEMPOL motion ( τ c,s , τ c,f ≤ 10 −7 s), over which hydrogen atom transfer and radical recombination reactions are also conducted.…”

“…17,18 Here, we address universal and particular properties of the confinement effect on the dynamics of the protein-associated solvent domain for different classes of soluble proteins by using the rotational correlation time (t c ; detection bandwidth, 10 À10 to 10 À7 s) of the electron paramagnetic resonance (EPR) spin probe, TEMPOL (B7 Å mean diameter), which is delimited to the vicinity of proteins in frozen aqueous solution by external, ice boundary confinement. 17,[19][20][21] Representative single-component EPR spectra of TEMPOL in the frozen mesodomain system, that illustrate the line shapes in the slow, intermediate and rapid tumbling regimes, are shown in Fig. S1 (simulations, parameters, Table S1, ESI †).…”