Slow Dynamics around a Protein and Its Coupling to Solvent

Solvent is essential for protein dynamics and function, but its role in regulating the dynamics remains debated. Here, we employ saturation transfer electron spin resonance (ST-ESR) to explore the issue and characterize the dynamics on a longer (from μs to s) time scale than has been extensively studied. We first demonstrate the reliability of ST-ESR by showing that the dynamical changeovers revealed in the spectra agree to liquid–liquid transition (LLT) in the state diagram of the glycerol/water system. Then, we utilize ST-ESR with four different probes to systematically map out the variation in local (site-specific) dynamics around a protein surface at subfreezing temperatures (180–240 K) in 10 mol % glycerol/water mixtures. At highly exposed sites, protein and solvent dynamics are coupled, whereas they deviate from each other when temperature is greater than LLT temperature (∼190 K) of the solvent. At less exposed sites, protein however exhibits a dynamic, which is distinct from the bulk solvent, throughout the temperature range studied. Dominant dynamic components are thus revealed, showing that (from low to high temperatures) the overall structural fluctuation, rotamer dynamics, and internal side-chain dynamics, in turn, dominate the temperature dependence of spin-label motions. The structural fluctuation component is relatively slow, collective, and independent of protein structural segments, which is thus inferred to a fundamental dynamic component intrinsic to protein. This study corroborates that bulk solvent plasticizes protein and facilitates rather than slaves protein dynamics

Concurrent Observation of Bulk and Protein Hydration Water by Spin-Label ESR under Nanoconfinement

Under nanoconfinement the formation of crystalline ice is suppressed, allowing the study of water dynamics at subfreezing temperatures. Here we report a temperature-dependent investigation (170–260 K) of the behavior of hydration water under nanoconfinement by ESR techniques. A 26-mer-long peptide and the Bax protein are studied. This study provides site-specific information about the different local hydrations concurrently present in the protein/peptide solution, enabling a decent comparison of the hydration moleculesthose that are buried inside, in contact with, and detached from the protein surface. Such a comparison is not possible without employing ESR under nanoconfinement. Though the confined bulk and surface hydrations behave differently, they both possess a transition similar to the reported fragile-to-strong crossover transition around 220 K. On the contrary, this transition is absent for the hydration near the buried sites of the protein. The activation energy determined under nanoconfinement is found to be lower in surface hydration than in bulk hydration. The protein structural flexibility, derived from the interspin distance distributions <i>P</i>(<i>r</i>) at different temperatures, is obtained by dipolar ESR spectroscopy. The <i>P</i>(<i>r</i>) result demonstrates that the structural flexibility is strongly correlated with the transition in the surface water, corroborating the origin of the protein dynamical transition at subfreezing temperatures

Revealing Structural Changes of Prion Protein during Conversion from α‑Helical Monomer to β‑Oligomers by Means of ESR and Nanochannel Encapsulation

Under nondenaturing neutral pH conditions, full-length mouse recombinant prion protein lacking the only disulfide bridge can spontaneously convert from an α-helical-dominant conformer (α-state) to a β-sheet-rich conformer (β-state), which then associates into β-oligomers, and the kinetics of this spontaneous conversion depends on the properties of the buffer used. The molecular details of this structural conversion have not been reported due to the difficulty of exploring big protein aggregates. We introduced spin probes into different structural segments (three helices and the loop between strand 1 and helix 1), and employed a combined approach of ESR spectroscopy and protein encapsulation in nanochannels to reveal local structural changes during the α-to-β transition. Nanochannels provide an environment in which prion protein molecules are isolated from each other, but the α-to-β transition can still occur. By measuring dipolar interactions between spin probes during the transition, we showed that helix 1 and helix 3 retained their helicity, while helix 2 unfolded to form an extended structure. Moreover, our pulsed ESR results allowed clear discrimination between the intra- and intermolecular distances between spin labeled residues in helix 2 in the β-oligomers, making it possible to demonstrate that the unfolded helix 2 segment lies at the association interface of the β-oligomers to form cross-β structure