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

Abstract

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 materials, 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 measurements 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.

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

“…Similarly, enzyme inactivation has been explained by a loss of secondary structure [46]. CD has also been used to confirm a maintained structure of peptides within pores at low temperatures (4 ºC) [100] and to prove an increased stability in the pores at elevated temperatures compared to free enzymes in the bulk [57,101,102]. In special cases, CD can be useful in the visible wavelength regions for proteins containing non-amino acid…”

“…Recently it has been shown that water molecules inside the nanochannels of mesoporous silica remain liquid way below normal freezing temperatures [156]. This observation has been utilized to study the relationship between biomolecules and surrounding hydrated water since it provides a more accurate biomolecular structure compared to cryostructures where the biomolecules attain a somewhat crystallized structure [100].…”

BAX-Induced Apoptosis Can Be Initiated through a Conformational Selection Mechanism

“…DEER experiments were carried out using the typical four-pulse constanttime DEER sequence as previously described (Huang et al, 2011;Pannier et al, 2000). The detection pulses were set to 32 and 16 ns for p and p/2 pulses, respectively, and pump frequency was set to approximately 70 MHz lower than the detection pulse frequency.…”

“…Advanced electron spin resonance (ESR) techniques (Borbat and Freed, 2007;Huang et al, 2011;Jeschke, 2012), including continuous-wave (cw) ESR and double electron-electron resonance (DEER), were used to reveal molecular details of the activation mechanisms of BAX. ESR with spin-labeling techniques has been established as a powerful tool for determining protein structure and topology.…”

Nanoscale Confinement and Fluorescence Effects of Bacterial Light Harvesting Complex LH2 in Mesoporous Silicas

“…A recent report has shown a further unique advantage of mesoporous materials in studying biological macromolecules at cryogenic temperatures, as nanopore-confined water remains in a liquid state even at subfreezing temperatures. 4 The remarkable progress in the development of mesoporous silica materials was initiated with the synthesis of MCM-41 first reported in 1992. 5 Different types of mesoporous silicas have since then been designed and prepared, 6−8 among which Santa Barbara Amorphous-15 (SBA-15) 9 and mesocellular silica foam (MCF) families 10 are of particular interest.…”