Spin-label ESR with nanochannels to improve the study of backbone dynamics and structural conformations of polypeptides

Abstract

Nanochannels of mesoporous silica materials were previously found useful for reducing the tumbling motion of encapsulated biomolecules while leaving the biomolecular structure undisturbed. Here we show that experiments of cw-ESR distance measurement in nano-confinement can benefit immediately from the above mentioned features of sufficiently slow molecular tumbling, enabling more accurate determination of interspin distances throughout the temperature range, from 200 to 300 K. A 26-residue prion protein peptide, which can fold into either a helical or hairpin structure, as well as its variants, are studied by using ESR. By comparing the spectra obtained in vitrified bulk solutions vs. mesopores, the spectra from the latter display typical slow-motional lineshapes, thereby enabling dipolar anisotropy to be unambiguously revealed throughout the temperature range, whereas the spectra from the former are dominated by the disordering of the side chain and the rotational tumbling of the peptide. The spectral changes regarding the two secondary structures in nano-confinement are found to show a strong correlation with the dynamic properties of the backbones. The effect of viscosity agent perturbation on the motion of an R1 nitroxide side chain, a commonly employed probe, could be substantial in a bulk solution condition, though it is absolutely absent in nanochannels. Under nano-confinement, the probe is proven sufficiently sensitive to the backbone motions. Overall, the distance distributions determined from the mesopore studies not only describe the conformational structures (by average distances), but also the backbone dynamics (by distribution widths) of the spin-labeled peptides.

ESR Study of Interfacial Hydration Layers of Polypeptides in Water-Filled Nanochannels and in Vitrified Bulk Solvents

“…The circular dichroism (CD) spectroscopy measurements for the n3-s and n3-d peptides as in the nanochannels versus in the bulk solvent (at 4°C and 25°C) were previously reported in Huang et al [12] and shown to resemble each other closely showing a typical β-hairpin structure (or an α-helical structure in the TFE/PB solvent). The results indicated that the secondary structures of the studied n3 peptide variants remain unchanged over the investigated experimental conditions [23].…”

“…[21] It is highly soluble and exhibits an intrinsic propensity to a β-hairpin conformation at neutral pH in the PB buffer (10 mM sodium phosphate, pH 6.5) or in pure water. [12] It was previously demonstrated that the n3 peptide folds into an α-helical conformation in a TFE(trifluoroethanol)/PB buffer. [12], [22] Two mutations of the n3 peptide were studied.…”

“…[12] It was previously demonstrated that the n3 peptide folds into an α-helical conformation in a TFE(trifluoroethanol)/PB buffer. [12], [22] Two mutations of the n3 peptide were studied. They were substituted with a cysteine at the 9th site and at both the 3rd and 9th sites, respectively, and were denoted as n3-s and n3-d (cf.…”

“…[9], [10] Built upon this progress, an approach combining ESR with nanochannels has emerged as a potentially useful tool for studying protein-water interactions; it demonstrated that the nanochannel of mesoporous materials was useful to maintain an amorphous state of solvents, which is required for studying protein structures and their dependence on water properties. [11], [12] Moreover, studying the biological surface water within nanochannels per se is also of fundamental importance in nature, since it is related to the origins of life. [13] Here, we report a carefully comparative investigation on the surface water molecules that directly interact with the biomolecules in nanochannels versus those for bulk solvents, and demonstrate that the surface hydration layer can be evidently recognized in the ESR spectroscopy.…”

Study of Protein Dynamics under Nanoconfinement by Spin-Label ESR: A Case of T4 Lysozyme Protein

“…[14][15][16] A variety of mesoporous materials were used together with ESR as a novel biophysical approach for probing dynamics on linear polypeptides. Previous studies showed that the nanochannels in mesoporous material provide well confined environment to immobilize the tumbling motions of the encapsulated molecules; the studied molecules include nitroxide-based radicals and several spin-labeled polypeptides with different lengths varying from 10 to 30 residues.…”