Identification of complex dynamic modes on prion protein peptides using multifrequency ESR with mesoporous materials

Abstract

Identifying protein dynamics is essential for studying protein function. However, the time-scale of dynamic modes varies over domains and segments of a protein. Here we describe an approach using multifrequency ESR with mesoporous materials for protein dynamics in confined nanospace that may mimic the crowded nature within a cell where proteins evolve to fold. While multifrequency ESR permits the separation of dynamic motions in different time-scales, we demonstrate its capability to capture dynamics can still be significantly enhanced by the encapsulation of nitroxide-labeled macromolecule into mesopores. Two mutants of a 26-residue prion protein peptide at temperatures from 2 to 27 °C are studied. The nanochannel provides the peptide with an ordered environment such that the global tumbling of peptide is slow, and 'frozen' on the ESR timescale. The local dynamic modes of the peptide in nanochannel are, therefore, distinctly reported on the spectra. The spectra of the peptide in β-hairpin vs.α-helical forms differ markedly, demonstrating the significant improvement of ESR spectroscopic capability due to our methodology. Such distinctly different spectral patterns between the two secondary structures of the peptide cannot be obtained from ESR studies in viscous aqueous solution. The dynamic modes on the peptide are thus unambiguously identified in our multifrequency experiments at the X- and Q-bands. Additionally, the multifrequency spectra for each mutant and temperature are simultaneously fitted to the rigorous models, e.g. the slowly-relaxing-local-structure model, for slow-motion ESR. Marked correlations are revealed and characterized quantitatively for the backbone flexibility between the β-hairpin and α-helical forms of the prion protein peptide. Confirmation of the slow collective dynamic modes extending across the β-hairpin is also provided through the spectral simulations.

Mesopores provide an amorphous state suitable for studying biomolecular structures at cryogenic temperatures

“…Sucrose and glycerol are commonly used as cryoprotectants in glass-forming solutions for low temperature experiments. At 275 K, the spectra of the n3-150R1 from the two aqueous viscous solutions exhibit a significantly faster tumbling motion than those from the mesopores, which is consistent with the previous finding (6). As the temperature was decreased to 200 K, both spectra were similar to the spectrum of the encapsulated n3-150R1, indicating that the tumbling motion of the peptide in the solution conditions is sufficiently slow and comparable to the tumbling motion in the mesopores.…”

“…This observation indicates the following: (i) the global tumbling of the peptides is sufficiently slow due to the nano-confinement effect within mesopores, (ii) the rotational correlation time is close to the lower bound of ESR time scale, and (iii) the local solvent structure, as reported in the spectra of the solvent-exposed site studied, changes insignificantly within the varied temperature range. It is consistent with several recent studies that a nanochannel is useful for slowing down the tumbling motions of molecules in solution within mesopores (6,22). The spectra of the two cryogenic temperatures (i.e., 70 and 50 K) were observed to be greater in the linewidth of each peak than those for 275 and 200 K. At lower temperatures, the rotation of the ring methyl groups of the spin label is slow, relative to the electron-proton hyperfine interactions (23,24).…”

“…As such, dipolar interaction between spins on a peptide is unambiguously obtained. It has also been demonstrated that the ESR spectral lineshape of the encapsulated n3 peptides is closely correlated with the backbone dynamics, rather than side-chain mobility and global tumbling (6). Presumably, the structural disordering (e.g., side-chain and backbone mobilities) of the n3 peptides in the two studied conditions are effectively arrested at such low cryogenic temperatures.…”

“…The encapsulation of spin-labeled samples into the mesoporous materials was prepared as previously described (6). The procedure was useful for incorporating spin-labeled biomolecules into the pores of mesoporous materials.…”

“…It is useful for studies of biocatalysis, biosensors (2-4), protein crystallization (5), and protein dynamics in confined nanochannels at high temperatures, such as room temperature (6). A key advantage of studying biomolecules entrapped within mesoporous materials is that biomolecular interactions (e.g., protein-protein interactions) can possibly be isolated/induced by tuning the structure of mesopores.…”