Structural Analyses of Designed α-Helix and ß-Sheet Peptide Nanofibers Using Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy and Introduction of Structure-Based Metal-Responsive Properties.

The 21-residue peptide α3, which is artificially designed and consists of three repeats of 7 residues, is known to rapidly assemble into the α-helix nanofiber. However, its molecular structure within the fiber has not yet been fully elucidated. Thus, we conducted a thorough investigation of the fiber's molecular structure using solid-state NMR and other techniques. The molecules were found to be primarily composed of the α-helix structure, with some regions near the C- and N-terminal adopting a 310-helix structure. Furthermore, it was discovered that ß-sheet hydrogen bonds were formed between the molecules at both ends. These intermolecular interactions caused the molecules to assemble parallelly in the same direction, forming helical fibers. In contrast, we designed two molecules, CaRP2 and ßKE, that can form ß-sheet intermolecular hydrogen bonds using the entire molecule instead of just the ends. Cryo-EM and other measurements confirmed that the nanofibers formed in a cross ß structure, albeit at a slow rate, with the formation times ranging from 1 to 42 days. To create peptide nanofibers that instantaneously respond to changes in the external environment, we designed several molecules (HDM1-3) based on α3 by introducing metal-binding sites. One of these molecules was found to be highly responsive to the addition of metal ions, inducing α-helix formation and simultaneously assembling into nanofibers. The nanofibers lost their structure upon removal of the metal ion. The change occurred promptly and was reversible, demonstrating that the intended level of responsiveness was attained.

Terahertz spectroscopy and solid-state density functional theory calculation of anthracene: effect of dispersion force on the vibrational modes.

The phonon modes of molecular crystals in the terahertz frequency region often feature delicately coupled inter- and intra-molecular vibrations. Recent advances in density functional theory such as DFT-D(*) have enabled accurate frequency calculation. However, the nature of normal modes has not been quantitatively discussed against experimental criteria such as isotope shift (IS) and correlation field splitting (CFS). Here, we report an analytical mode-decoupling method that allows for the decomposition of a normal mode of interest into intermolecular translation, libration, and intramolecular vibrational motions. We show an application of this method using the crystalline anthracene system as an example. The relationship between the experimentally obtained IS and the IS obtained by PBE-D(*) simulation indicates that two distinctive regions exist. Region I is associated with a pure intermolecular translation, whereas region II features coupled intramolecular vibrations that are further coupled by a weak intermolecular translation. We find that the PBE-D(*) data show excellent agreement with the experimental data in terms of IS and CFS in region II; however, PBE-D(*) produces significant deviations in IS in region I where strong coupling between inter- and intra-molecular vibrations contributes to normal modes. The result of this analysis is expected to facilitate future improvement of DFT-D(*).

Assessment of density functional theory to calculate the phase transition pressure of ice.

To assess the accuracy of density functional theory (DFT) methods in describing hydrogen bonding in condensed phases, we benchmarked their performance in describing phase transitions among different phases of ice. We performed DFT calculations of ice for phases Ih, II, III, VI and VII using BLYP, PW91, PBE, PBE-D, PBEsol, B3LYP, PBE0, and PBE0-D, and compared the calculated phase transition pressures between Ih-II, Ih-III, II-VI, and VI-VII with the 0 K experimental values of Whalley [J. Chem. Phys., 1984, 81, 4087]. From the geometry optimization of many different candidates, we found that the most stable proton orientation as well as the phase transition pressure does not show much functional dependence for the generalized gradient approximation and hybrid functionals. Although all these methods overestimated the phase transition pressure, the addition of van der Waals (vdW) correction using PBE-D and PBE0-D reduced the transition pressure and improved the agreement for Ih-II. On the other hand, energy ordering between VI and VII reversed and gave an unphysical negative transition pressure. Binding energy profiles of a few conformations of water dimers were calculated to understand the improvement for certain transitions and failures for others with the vdW correction. We conclude that vdW dispersion forces must be considered to accurately describe the hydrogen bond in many different phases of ice, but the simple addition of the R(-6) term with a small basis set tends to over stabilize certain geometries giving unphysical ordering in the high density phases.

Low-frequency dynamics of bacteriorhodopsin studied by terahertz time-domain spectroscopy.

We measured low-frequency spectra of bacteriorhodopsin (BR) by terahertz (THz; 1 THz approximately = 33 cm(-1)) time-domain spectroscopy. Both the absorption coefficient and the refractive index were obtained simultaneously in the THz frequency region. The dependence of the THz spectra on hydration and temperature was studied in detail. We defined the reduced absorption cross-section (RACS) which was estimated from the product of the absorption coefficient and the refractive index. RACS exhibited power-law behavior in the frequency region from 7 cm(-1) to 26 cm(-1), and we investigated the hydration and temperature dependence of spectral features such as the magnitude of RACS and the exponent in the power law for RACS. For the dried BR sample, the observed spectral dependence on hydration and temperature suggests that anharmonic coupling between low-frequency modes was relatively weak. For the hydrated BR sample, the temperature dependence of the spectral features was similar to that of the dried sample in the temperature range from -100 degrees C to around -40 degrees C. The THz spectra of the hydrated BR markedly changed at about -40 degrees C, similar to an inelastic neutron scattering experiment which indicates that the mean-square displacement of BR substantially changes at -40 degrees C. We discuss the relationship between the THz spectra, the inelastic neutron scattering spectra, and the function of BR.

Terahertz time-domain spectroscopy of poly-L-lysine.

Poly-L-lysine is known to have three different secondary structures depending on solvent conditions because of its flexible nature. In previous work (Kambara et al., Phys Chem Chem Phys 2008, 10, 5042-5044), we observed two different types of structural changes in poly-L-lysine. In the present study, we investigated the low-frequency spectrum of poly-L-lysine with a beta-sheet structure in the solid state by terahertz time-domain spectroscopy. On the basis of this spectroscopic analysis, we found that the low-frequency dynamics differed from those of other polypeptides. Furthermore, we performed powder X-ray diffraction measurement on poly-L-lysine, which was found to be highly amorphous compared with other polypeptides.

Structural changes of poly-L-lysine in solution and lyophilized form.

Two structural changes of poly-L-lysine have been studied by various spectroscopic techniques; one is a structural change of a random coil sample in solution to a mixture of alpha-helix and beta-sheet during rapid freezing in the lyophilizing process, and the other is a pressure-induced structural change from an alpha-helix to a beta-sheet structure for a lyophilized sample.

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification.

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel. This includes automated implementation of negative design by explicitly requiring higher free energy of the denatured state. The designed sequences do not have significant similarity to those of any natural proteins. The NMR and CD spectroscopic data indicated that one designed sequence has a well-defined three-dimensional structure as well as alpha-helical content consistent with the target.