Mesodomain and Protein-Associated Solvent Phases with Temperature-Tunable (200–265 K) Dynamics Surround Ethanolamine Ammonia-Lyase in Globally Polycrystalline Aqueous Solution Containing Dimethyl Sulfoxide

Abstract

Electron paramagnetic resonance (EPR) spectroscopy of the spin probe, TEMPOL, is used to resolve solvent phases that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium at low-temperature (T) in frozen, globally polycrystalline aqueous solution, and to report on the T-dependence of their detectably rigid and fluid states. EAL plays a role in human gut microbiome-based disease conditions, and physical-chemical studies provide insight into protein structure and mechanism, toward potential therapeutics. Temperature dependences of the rotational correlation times (τc; detection range, 10−11≤τc ≤10−7 s) and corresponding weights of TEMPOL tumbling components from 200 to 265 K in the presence of EAL, are measured in two frozen systems: (1) water-only, and (2) 1% v/v dimethylsulfoxide (DMSO). In the water-only condition, a protein-vicinal solvent component detectably fluidizes at 230 K, and melts the surrounding ice-crystalline region with increasing T, creating a bounded, relatively high-viscosity aqueous solvent domain, up to 265 K. In the EAL, 1% DMSO condition, two distinct concentric solvent phases are resolved around EAL: protein-associated domain (PAD) and mesodomain. The DMSO-aqueous mesodomain fluidizes at 200 K, followed by PAD fluidization at 210 K. The interphase-dynamical coupling is consistent with the spatial arrangement and significant contact areas of the phases, indicated by the experimentally-determined mean volume ratio, V(mesodomain):V(PAD):V(protein) = 0.5:0.3:1.0. The results provide a rationale for native chemical reactions of EAL at T<250 K, and an advance toward precise control of solvent dynamics as a tunable parameter for quantifying the coupling between solvent and protein fluctuations and chemical reaction steps in EAL, and other enzymes.

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

“…The assignment of the PAD solvent phase was based on the direct dependence of its volume on the varied EAL concentration, with no PAD component observed in the absence of EAL. 14 The mesodomain solvent phase assignment reflected the near-identical T dependences of TEMPOL mobility in the absence and presence of EAL. 14 The X-band EPR spin probe approach has previously been used to characterize the structure and dynamics of mesodomains in frozen, bulk polycrystalline aqueous sucrose, 18 aqueous glycerol, 21 and subphases in other solvent mixtures, 22 in the absence of protein.…”

“…14 The mesodomain solvent phase assignment reflected the near-identical T dependences of TEMPOL mobility in the absence and presence of EAL. 14 The X-band EPR spin probe approach has previously been used to characterize the structure and dynamics of mesodomains in frozen, bulk polycrystalline aqueous sucrose, 18 aqueous glycerol, 21 and subphases in other solvent mixtures, 22 in the absence of protein. TEMPOL rotational motion leads to averaging of the unpaired electron (S = 1/2) g and electron− 14 N (I = 1) dipolar hyperfine anisotropies and a consequent narrowing of the EPR line shape, 13 which is quantified by the rotational correlation time (τ c ) obtained from spectral simulations.…”

“…TEMPOL is also excluded from the ice domains. 14,18 As T descends, the cosolvent concentration in the mesodomain is elevated to the maximum freeze concentration, which corresponds to the eutectic point in the melting temperature composition dependence. 15 For aqueous DMSO solutions, the maximum freeze concentration is 65% v/v.…”

“…19 The aqueous− DMSO in the low-T mesodomain forms a high-viscosity (η) fluid in the T range of 195−265 K. 20 Values of η of 14−340 cP over 263−218 K and a calorimetrically determined glasstransition temperature of T g = 145 K are reported for bulk aqueous 65% v/v DMSO solutions. 20 Previous measurements of the dependence of TEMPOL spin probe rotational mobility in frozen solutions on the presence and absence of EAL and added 1% v/v DMSO resolved two solvent components that surround the protein: 14 (1) a protein-associated domain (PAD; proposed to correspond to the protein hydration layer), characterized by relatively low TEMPOL mobility, and…”

“…Continuous-wave EPR measurements were performed by using a Bruker E500 ElexSys EPR spectrometer and a Bruker ER4123SHQE X-band cavity resonator, as described. 14 EPR acquisition parameters: microwave frequency, 9.45 GHz; microwave power, 0.2 mW; magnetic field modulation, 0.2 mT; modulation frequency, 100 kHz; acquisition number, four to eight spectra were averaged at each T value.…”