Ohki Kambara

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.

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.

Temperature and hydration dependence of low-frequency spectra of poly-L-glutamic acid with different secondary structures studied by terahertz time-domain spectroscopy

We have investigated low-frequency spectra of poly-L-glutamic acid (polyE) in the powder state by terahertz time-domain spectroscopy (THz-TDS). Samples with three different secondary structures (α-helix, β-sheet, and random-coil) and different chain lengths were prepared to investigate the dependence of the THz spectra on temperature and hydration. The temperature dependence of the THz absorption spectra clearly shows that polyE, regardless of its secondary structure, undergoes dynamical transition between 190 and 240 K. We have estimated the apparent activation energy and transition temperature by phenomenological spectral analysis. We also have estimated the effective dipole moment of the amino acid residue from the real part of the dielectric permittivity at zero frequency. Both results show that the transition temperature is lower when the secondary structure undergoes a transition from a random-coil structure to an α-helix or β-sheet structure. Furthermore, both hydrating water molecules and peptide hydrogen bonds contribute to induce anharmonicity in the low-frequency vibrational motions. Meanwhile, hydration, not peptide hydrogen bonds, is crucial for the dynamical transition to occur because the onset of anharmonicity was observed only when the polypeptide is hydrated. An apparent intermolecular vibrational mode in the β-sheet structure, which suggests a highly ordered structure in the sample, did not exhibit anharmonicity at the tested temperatures and humidity levels. This result suggests that short-range or inter-strand hydrogen bonds of the α-helix or low-ordered β-sheet structures gave rise to the lower transition temperatures and the smaller effective activation energies compared with those of the random-coil structure.

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.

Structural fluctuation of proteins revealed by terahertz time-domain spectroscopy

We have measured spectra of the absorption coefficient and refractive index of hen egg white lysozyme in the wavenumber region from 7 cm −1 to 50 cm −1 by terahertz (THz) time-domain spectroscopy. From comparison with the results of the inelastic neutron scattering experiment it is concluded that analysis of the THz spectra provides information on the vibrational density of states. We studied temperature dependence of the THz spectra as well as hydration effect on them to discuss structural fluctuation of the protein. We observed dynamical transition at around 210 K for lysozyme.

Low-frequency dynamics of proteins and aqueous solutions studied by terahertz time-domain spectroscopy

We studied low-frequency spectra of hydration water molecules around the hydrophobic probe in an aqueous solution by using tetraalkylammonium cation as a probe and terahertz time-domain spectroscopic technique. The phenomenon, called dynamical transition, has been known to be universally observed among proteins and polypeptides. In this work we investigated temperature and hydration dependence of low-frequency dynamics to clarify relationships between the dynamical transition and protein structures, and its functional states. We also mention general behaviors of the lowfrequency spectra of globular proteins.

Terahertz time-domain spectroscopy on proteins, peptides, and small biological molecules

Absorption coefficients and refractive indices of biologically important macromolecules such as lysozyme and myoglobin and small molecules such as adenosine triphosphate (ATP) have been obtained in the low-frequency region by terahertz time-domain spectroscopy (THz-TDS) to understand molecular dynamics and interactions of these molecules.

Low-frequency dynamics of hydrogen-bonding small organic molecules

Single frequency coherent terahertz-wave generation method is used to obtain low-frequency spectra of the hydrogen bonding organic small molecules. Transmission spectrum of each sample show several peaks in this region. On the other hand, ab initio frequency calculations are performed for each molecule by CRYSTAL06 package. Both experimental results and theoretical calculations are compared each other.

Low-frequency spectra of amino acids and peptides probed by terahertz time-domain spectroscopy

Low-frequency spectra of amino acids and peptides of L-alanine and glycine were analyzed from the power law behavior of its absorption cross section. For amino acids the exponent around two were observed and interpreted as a collection of harmonic vibrations. However for peptides with longer chain i.e. (Gly)4 and (L-ala)5 exponents were reduced to 1.43 and 1.34, respectively. This behavior shows chain length dependence of the low frequency dynamics.

Temperature and hydration effect on the low-frequency dynamics of proteins

Absorption coefficient and refractive index of hen egg white lysozyme have been obtained in the frequency region of 3–50 cm−1 to estimate reduced absorption cross section (RACS). Experimental conditions such as hydration and temperature were varied, and we found by using RACS analysis that high temperature and high hydration condition is needed for proteins to be set in the functional state.