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

Abstract

There is considerable evidence for the essential role of surface water in protein function and structure. However, it is unclear to what extent the hydration water and protein are coupled and interact with each other. Here, we show by ESR experiments (cw, DEER, ESEEM, and ESE techniques) with spin-labeling and nanoconfinement techniques that the vitrified hydration layers can be evidently recognized in the ESR spectra, providing nanoscale understanding for the biological interfacial water. Two peptides of different secondary structures and lengths are studied in vitrified bulk solvents and in water-filled nanochannels of different pore diameter (6.1∼7.6 nm). The existence of surface hydration and bulk shells are demonstrated. Water in the immediate vicinity of the nitroxide label (within the van der Waals contacts, ∼0.35 nm) at the water-peptide interface is verified to be non-crystalline at 50 K, and the water accessibility changes little with the nanochannel dimension. Nevertheless, this water accessibility for the nanochannel cases is only half the value for the bulk solvent, even though the peptide structures remain largely the same as those immersed in the bulk solvents. On the other hand, the hydration density in the range of ∼2 nm from the nitroxide spin increases substantially with decreasing pore size, as the density for the largest pore size (7.6 nm) is comparable to that for the bulk solvent. The results demonstrate that while the peptides are confined but structurally unaltered in the nanochannels, their surrounding water exhibits density heterogeneity along the peptide surface normal. The causes and implications, especially those involving the interactions between the first hydration water and peptides, of these observations are discussed. Spin-label ESR techniques are proven useful for studying the structure and influences of interfacial hydration.

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

“…Enzyme stability has been shown to be related to an increased hydration inside the pores that stabilize the enzyme structure based on measurements with PPC and DSC (section 7.2) [142]. Density differences in water around poly peptides in SBA-15 and consequently varied hydration has been shown using spin-labeled electron spin resonance (ESR) [157]. Therefore, the hydration of the active site of an enzyme will depend on the distance to the pore wall and the orientation of the enzyme inside the pores.…”

Identifying Protein Conformational Dynamics Using Spin‐label ESR

“…Bulk water and surface hydration of spin-labeled biomolecules were shown to stay non-crystalline and vitrifieda ts ub-freezing temperatures when encapsulated in nanochannels of mesoporous materials or silica gel. [84][85][86][87][88] Making use of this nanoconfinemente ffect, spin-label ESR has been improved with mesoporousm aterials such that protein and water dynamics can be studied by ESR at low temperatures withoutc ryoprotective additives. [89,90] As the global tumbling motions of ap rotein/probe are largely slowed down by nanoconfinement, ESR spectra become sensitive to dynamics related to protein backbone and internal motions even at room temperatures (Fig-ure 5A).…”

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

“…This finding has opened up new avenues for studying water dynamics within the no man's land (150−235 K) where bulk water is crystalline. [10][11][12][13] Despite the variety of applications, a comprehensive investigation and characterization of the dynamics present in a nanoconfined protein remains to be explored. To gain more knowledge about the immobilization/encapsulation process, it is crucial to reveal details of how protein structure and dynamics are affected by the microenvironment inside the nanochannels.…”

“…11,12 Taken together, advantages of studying biomolecules under nanoconfinement by ESR include: i) reduced sensitivity of ESR spectral lineshape to the global tumbling motions of a biomolecule, ii) improved spectral sensitivity to LBF along a segment of secondary structure, and iii) ESR measurement at higher temperatures (e.g. 300 K) without the use of viscosity reagents.…”