Self-Assembly of Polydeoxyadenylic Acid Studied at the Single-Molecule Level

The investigation on the self-assembly of polydeoxyadenylic acid (poly(dA)) is highly important to fully understand its biological function and for its application in the field of nanotechnology. Using the fluorescence resonance energy transfer (FRET) technique, we report investigations for the self-assembly of adenine oligomers induced by pH and coralyne binding at the single-molecule level and in the bulk phase. Results presented here show that A-motif 1 (Alexa488-5′-(dA)20-3′-Cy5-5′-(dA)20-3′-Alexa488) forms the wire-type duplex at acidic pH, whereas the same conformation of A-motif 2 (Alexa488-5′-(dA)20-3′-Cy5-3′-(dA)20-5′-Alexa488) is induced by coralyne binding at neutral pH. These results indicate that poly(dA) at acidic pH forms a right-handed helical duplex with parallel-mannered chains, whereas the coralyne–poly(dA) binding induces a stable antiparallel duplex. Furthermore, we found that the antiparallel duplex of poly(dA) formed by coralyne binding has a rather extended and less twisted structure as compared to the parallel duplex of poly(dA) formed at acidic pH. On the other hand, from dilution experiments, we found that the parallel duplex formed at acidic pH is converted to “S-form”, which has the single-stranded structure with short intramolecular double-stranded regions formed by intramolecular A:A base pairing, while the A-motif–coralyne assembly is dissociated into single strands below a certain concentration. The formation of S-form with a short intramolecular double-stranded region formed at acidic pH and very low concentration is confirmed by the quantitative analysis of FCS curve to measure the hydrodynamic radius of a molecule

Folding Dynamics of Cytochrome <i>c</i> Using Pulse Radiolysis

Pulse radiolysis is a powerful method to realize real-time observation of various redox processes, which induces various structural and functional changes occurring in biological systems. However, its application has been mainly limited to studies of the redox reactions of rather smaller biological systems such as DNA because of an undesired reaction due to various free radicals generated by pulse radiolysis. For application of pulse radiolysis to generate plenty of redox reactions of biological systems, selective redox reactions induced by electron pulses have to be developed. In this study, we report that in the presence of the high concentration of the denaturant, guanidine HCl (GdHCl), the selective reduction of the oxidized cytochrome <i>c</i> (Cyt <i>c</i>) takes place in time scales of a few microseconds by the electron transfer from the guanidine radical that is formed by the fast reaction of e_aq^– with GdHCl, consequently leading to folding kinetics of Cyt <i>c</i>. By providing insight into the folding dynamics of Cyt <i>c</i>, we show that the pulse radiolysis technique can be used to track the folding dynamics of various biomolecules in the presence of a denaturant including GdHCl

Interaction of G‑Quadruplex with RecA Protein Studied in Bulk Phase and at the Single-Molecule Level

As in the human genome there are numerous repeat DNA sequences to adopt into non-B DNA structures such as hairpin, triplex, Z-DNA, G-quadruplex, and so on, an understanding of the interaction between DNA repair proteins and a non-B DNA forming sequence is very important. In this regard, the interaction between RecA protein and human telomeric 5′-TAGGG-(TTAGGG)₃-TT-3′ sequence and the G-quadruplex formed from this sequence has been investigated in bulk phase and at the single-molecule level. The RecA@ssDNA filament, which is formed by the interaction between RecA protein and a G-rich sequence, was dissociated by the addition of K⁺ ions, and the dissociated G-rich sequence was quickly folded to a G-quadruplex structure, indicating that the G-quadruplex structure is more favorable than the RecA@ssDNA filament in the presence of K⁺ ions. In addition, we demonstrate that the conformation of the G-quadruplex, which is heterogeneous in the absence of RecA, converged to the specific G-quadruplex with one double-chain-reversal loop upon association of RecA protein

pH-Induced Intramolecular Folding Dynamics of i-Motif DNA

Using the combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) technique, we investigate the mechanism and dynamics of the pH-induced conformational change of i-motif DNA in the bulk phases and at the single-molecule level. Despite numerous studies on i-motif that is formed from cytosine (C)-rich strand at slightly acidic pH, its detailed conformational dynamics have been rarely reported. Using the FRET technique to provide valuable information on the structure of biomolecules such as a protein and DNA, we clearly show that the partially folded species as well as the single-stranded structure coexist at neutral pH, supporting that the partially folded species may exist substantially in vivo and play an important role in a process of gene expression. By measuring the FCS curves of i-motif, we observed the gradual decrease of the diffusion coefficient of i-motif with increasing pH. The quantitative analysis of FCS curves supports that the gradual decrease of diffusion coefficient (D) associated with the conformational change of i-motif is not only due to the change in the intermolecular interaction between i-motif and solvent accompanied by the increase of pH but also due to the change of the shape of DNA. Furthermore, FCS analysis showed that the intrachain contact formation and dissociation for i-motif are 5–10 times faster than that for the open form. The fast dynamics of i-motif with a compact tetraplex is due to the intrinsic conformational changes at the fluorescent site including the motion of alkyl chain connecting the dye to DNA, whereas the slow intrachain contact formation observed from the open form is due to the DNA motion corresponding to an early stage interaction in the folding process of the unstructured open form

Detection of Structural Changes upon One-Electron Oxidation and Reduction of Stilbene Derivatives by Time-Resolved Resonance Raman Spectroscopy during Pulse Radiolysis and Theoretical Calculations

Stilbene (St) derivatives have been investigated for many years because of their interesting photochemical reactions such as cis–trans isomerization in the excited states and charged states and their relation to poly­(<i>p</i>-phenylenevinylene)­s. To clarify their charged state properties, structural information is indispensable. In the present study, radical cations and radical anions of St derivatives were investigated by radiation chemical methods. Absorption spectra of radical ion states were obtained by transient absorption measurements during pulse radiolysis; theoretical calculations that included the solvent effect afforded reasonable assignments. The variation in the peak position was explained by using HOMO and LUMO energy levels. Structural changes upon one-electron oxidation and reduction were detected by time-resolved resonance Raman measurements during pulse radiolysis. Significant downshifts were observed with the CC stretching mode of the ethylenic groups, indicative of the decrease in the bonding order. It was confirmed that the downshifts observed with reduction were larger than those with oxidation. On the other hand, the downshift caused by oxidation depends significantly on the electron-donating or electron-withdrawing nature of the substituents

Structural Study of Various Substituted Biphenyls and Their Radical Anions Based on Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis

The structures of various <i>para</i>-substituted biphenyls (Bp-X; X = −OH, −OCH₃, −CH₃, −H, −CONH₂, −COOH, and −CN) and their radical anions (Bp-X^•–) were investigated by time-resolved resonance Raman spectroscopy combined with pulse radiolysis. The inter-ring C1–C1′ stretching modes (ν₆) of Bp-X were observed at ∼1285 cm^–1, whereas the ν₆ modes of Bp-X^•– with an electron-donating or -withdrawing substituent were significantly up-shifted. The difference (Δ<i>f</i>) between the ν₆ frequencies of Bp-X and Bp-X^•– showed a significant dependence on the electron affinity of the substituent and exhibited a correlation with the Hammett substituent constants (σ_p). In contrast to Bp-H^•– with a planar geometry, the theoretical and experimental results reveal that all Bp-X^•– with an electron-donating or -withdrawing substituent have a slightly twisted structure. The twisted structure of Bp-X^•– is due to the localization of the unpaired electron and negative charge density on one phenyl moiety in Bp-X^•–

Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis

The oxidation of guanine (G) is studied by using transient absorption and time-resolved resonance Raman spectroscopies combined with pulse radiolysis. The transient absorption spectral change demonstrates that the neutral radical of G (G^•(−H⁺)), generated by the deprotonation of G radical cation (G^•+), is rapidly converted to other G radical species. The formation of this species shows the pH dependence, suggesting that it is the G radical cation (G^•+)′ formed from the protonation at the N7 of G^•(−H⁺). On one hand, most Raman bands of (G^•+)′ are up-shifted relative to those of G, indicating the increase in the bonding order of pyrimidine (Pyr) and imidazole rings. The (G^•+)′ exhibits the characteristic CO stretching mode at ∼1266 cm^–1 corresponding to a C–O single bond, indicating that the unpaired electron in (G^•+)′ is localized on the oxygen of the Pyr ring

Length and Charge of the N‑terminus Regulate the Lifetime of the Signaling State of Photoactive Yellow Protein

Photoactive yellow protein (PYP) is one of the most extensively studied photoreceptors. Nevertheless, the role of the N-terminus in the photocycle and structural transitions is still elusive. Here, we attached additional amino acids to the N-terminus of PYP and investigated the effect of the length and charge of additional N-terminal residues using circular dichroism, two-dimensional nuclear magnetic resonance (2D-NMR), transient absorption (TA), and transient grating (TG) spectroscopic techniques. TA experiments showed that, except for negatively charged residues (5D-PYP), additional N-terminal residues of PYP generally enable faster dark recovery from the putative signaling state (pB2) to the ground state (pG). TG data showed that although the degree of structural changes can be controlled by adjusting specific amino acid residues in the extended N-terminus of N-terminal extended PYPs (NE-PYPs), the dark recovery times of wt-PYP and NE-PYPs, except for 5D-PYP, are independent of the structural differences between pG and pB2 states. These results demonstrate that the recovery time and the degree of structural change can be regulated by controlling the length and sequence of N-terminal residues of PYP. The findings in this study emphasize the need for careful attention to the remaining amino acid residues when designing recombinant proteins for genetic engineering purposes

Ultrafast Structural Dynamics of the Photocleavage of Protein Hybrid Nanoparticles

Protein-coated gold nanoparticles in suspension are excited by intense laser pulses to mimic the light-induced effect on biomolecules that occur in photothermal laser therapy with nanoparticles as photosensitizer. Ultrafast X-ray scattering employed to access the nanoscale structural modifications of the protein–nanoparticle hybrid reveals that the protein shell is expelled as a whole without denaturation at a laser fluence that coincides with the bubble formation threshold. In this ultrafast heating mediated by the nanoparticles, time-resolved scattering data show that proteins are not denatured in terms of secondary structure even at much higher temperatures than the static thermal denaturation temperature, probably because time is too short for the proteins to unfold and the temperature stimulus has vanished before this motion sets in. Consequently the laser pulse length has a strong influence on whether the end result is the ligand detachment (for example drug delivery) or biomaterial degradation

Visualizing Heterogeneous Protein Conformations with Multi-Tilt Nanoparticle-Aided Cryo-Electron Microscopy Sampling

Obtaining the heterogeneous conformation of small proteins is important for understanding their biological role, but it is still challenging. Here, we developed a multi-tilt nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) technique that enables the observation of heterogeneous conformations of small proteins and applied it to calmodulin. By imaging the proteins labeled by two gold nanoparticles at multiple tilt angles and analyzing the projected positions of the nanoparticles, the distributions of 3D interparticle distances were obtained. From the measured distance distributions, the conformational changes associated with Ca2+ binding and salt concentration were determined. MT-NACS was also used to track the structural change accompanied by the interaction between amyloid-beta and calmodulin, which has never been observed experimentally. This work offers an alternative platform for studying the functional flexibility of small proteins