Effects of Anisotropic Nanoconfinement on Rotational Dynamics of Biomolecules: An Electron Spin Resonance Study

The development of nanostructured materials for next-generation nanodevice technologies requires a better understanding of dynamics of the objects as confined in nanospace. Here, we characterize the rotational dynamics of a long (14-residue) proline-based peptide (approximately 4 nm in length) under anisotropic nanoconfinement using spin-labeling CW/pulsed ESR techniques as well as spectral simulations. We show by pulsed ESR experiments that the conformations of the peptide in several different nanochannels and a bulk solvent are retained. Parameters characterizing the dynamics of the peptide regarding temperature (200–300 K) and nanoconfinement are determined from nonlinear least-squares fits of theoretical calculations to the multifrequency experimental spectra. Remarkably, we find that this long helical peptide undergoes a large degree of rotational anisotropy and orientational ordering inside the nanochannels, but not in the bulk solvent. The rotational anisotropy of the helical peptide barely changes with the nanoconfinement effects and remains substantial, as the nanochannel diameter is varied from 6.1 to 7.1 and 7.6 nm. This finding is advantageous for addressing purposes of anisotropic nanoconfinement and for advancing our understanding of the rotational dynamics of nano-objects as confined deeply inside the nanostructures of materials

Side-Chain Packing Interactions Stabilize an Intermediate of BAX Protein against Chemical and Thermal Denaturation

Bcl-2-associated X (BAX) protein plays a gatekeeper role in transmitting apoptotic signaling from cytosol to mitochondria. However, little is known about its stability. This study reports a comprehensive investigation on the stability of BAX using spin-label ESR, CD, and ThermoFluor methods. Point mutations covering all of the nine helices of BAX were prepared. ESR study shows that BAX can be divided into two structural regions, each responding differently to the presence of guanidine hydrochloride (GdnHCl). The N-terminal region (helices 1–3) is denatured in 6 M GdnHCl, whereas the C-terminal region (helices 4–9) is resistant to the denaturing effects. The far-UV CD spectra show an appreciable amount of helical content of BAX at high temperatures. The magnitude of the near-UV CD signal is increased with increasing temperature in either 0 or 6 M GdnHCl, indicating an enhancement of aromatic side-chain packing in the C-terminal region. Taken together with ThermoFluor results, we show that a core interior, wherein aromatic interactions are highly involved, within the C-terminal region plays an important role in stabilizing BAX against the denaturing effects. Collectively, we report a highly stable, indestructible intermediate state of BAX. Side-chain packing interactions are shown to be the major stabilizing force in determining BAX structure