Mesopores provide an amorphous state. Here, we report an improvement in ESR spectral sensitivity to local backbone dynamics of a protein by a methodology that performs ESR measurement when the protein is confined in the nanochannels of a mesoporous material. An extensive set of ESR data, which includes the spectra of a nitroxide-based side chain from buried and solvent-exposed sites of a T4 lysozyme (T4L) protein, were obtained over a range of temperatures, 200−300 K, to explore the dynamics of T4L under nanoconfinement. Spectra were simulated by performing theoretical fits to the data using the microscopic ordering with macroscopic disordering model. Two principle dynamic modes, which differ in mobility and ordering, are required to account for the spectra at temperatures >240 K. We show that the mobile one correlates only with the local backbone dynamics of buried sites, whereas the other reflects the difference in local hydration dynamics between the labeling sites in T4L. The assignment of the mobile component is supported by the X-ray crystallography data of T4L. Collectively, this study has demonstrated the validity of such a methodology for improving ESR sensitivity to buried sites in a protein.

Journal of Physical Chemistry B, 116 (2017) 4355-4363

We investigate the hydration dynamics of the bulk water, surface water, and internal water of a protein by SDSL-ESR experiments measured under nanoconfinement. It demonstrates that the structural flexibility of a protein is strongly correlated with the transition in the surface water, corroborating the origin of the protein dynamical transition at subfreezing temperatures.

ACS Chemical Biology, 10 (2015) 493-501

Langmuir, 29 (2013) 13865-13872

PLOS one, 8 (2013) e68264-13

Physical Chemistry Chemical Physics, 13 (2011) 17521-17531

In nano-confinements, aqueous solutions can be found to remain in a liquid state at subfreezing temperatures. The finding provides a means of entering into previously inaccessible temperature regions for studying the dynamics and structure of bulk liquid. Here we show that studying biomolecular structures in nano-confinements improves the accuracy of cryostructures and provides better insight into the relationship between hydration water and biomolecules. Synthetic prion protein peptides are studied in two experimental conditions: (i) in confined nanochannels within mesoporous mate- rials, and (ii) in vitrified bulk solvents, with a temperature range of 50–275 K, using cw/pulse ESR techniques. A large inhomogeneous lineshape broadening is only observed for the spectra from the vitrified bulk solvent below 70 K, suggesting a possible peptide clustering in the solution. The spin-counting and distance measure- ments by DEER-ESR provide further evidence that peptides are dispersed homogeneously in mesopores but heterogeneously in vitrified solvents wherein the biomolecular structure is disturbed due to heterogeneity in the bulk solvent structure. Our study demonstrates that the nanospace within mesoporous materials provides an amorphous environment that is better than vitrified bulk solvent for studying biostructures at cryogenic temperatures.

Journal of Physical Chemistry C, 116 (2012) 19798-19806

Proceedings of the National Academy of Sciences (PNAS), 108 (2011) 14145-14150