Excited states of GFP chromophore and active site studied by the SAC-CI method: effect of protein-environment and mutations

Excited states of fluorescent proteins were studied using symmetry-adapted cluster-configuration interaction (SAC-CI) method. Protein-environmental effect on the excitation and fluorescence energies was investigated. In green fluorescent protein (GFP), the overall protein-environmental effect on the first excitation energy is not significant. However, glutamine (Glu) 94 and arginine (Arg96) have the red-shift contribution as reported in a previous study (Laino et al., Chem Phys 2004, 298, 17). The excited states of GFP active site (GFP-W22-Ser205-Glu222-Ser65) were also calculated. Such large-scale SAC-CI calculations were performed with an improved code containing a new algorithm for the perturbation selection. The SAC-CI results indicate that a charge-transfer state locates at 4.19 eV, which could be related to the channel of the photochemistry as indicated in a previous experimental study. We also studied the excitation and fluorescence energies of blue fluorescent protein, cyan fluorescent protein, and Y66F. The SAC-CI results are very close to the experimental ones. The protonation state of blue fluorescent protein was determined. Conformation of cyan fluorescent protein indicated by the present calculation agrees to the experimentally observed structure.     

Photoinduced intramolecular electron transfer and triplet energy transfer in a steroid-linked norbornadiene-carbazole dyad

Bichromophoric compound 3 beta-((2-(methoxycarbonyl)bicyclo[2.2.1]hepta-2,5-diene-3-yl)carboxy)androst-5-en-17 beta-yl-[2-(N-carbazolyl)acetate] (NBD-S-CZ) was synthesized and its photochemistry was examined by fluorescence quenching, flash photolysis, and chemically induced dynamic nuclear polarization (CIDNP) methods. Fluorescence quenching measurements show that intramolecular electron transfer from the singlet excited state of the carbazole to the norbornadiene group in NBD-S-CZ occurs with an efficiency (Phi SET) of about 14 % and rate constant (kSET) of about 1.6 x 10(7) s-1. Phosphorescence and flash photolysis studies reveal that intramolecular triplet energy transfer and electron transfer from the triplet carbazole to the norbornadiene group proceed with an efficiency (TET + TT) of about 52 % and rate constant (kTET + kTT) of about 3.3 x 10(5) s-1. Upon selective excitation of the carbazole chromophore, nuclear polarization is detected for protons of the norbornadiene group (emission) and its quadricyclane isomer (enhanced absorption); this suggests that the isomerization of the norbornadiene group to the quadricyclane proceeds by a radical-ion pair recombination mechanism in addition to intramolecular triplet sensitization. The long-distance intramolecular triplet energy transfer and electron transfers starting both from the singlet and triplet excited states are proposed to proceed by a through-bond mechanism.     

Electronic and vibronic contributions to two-photon absorption in donor-acceptor-donor squaraine chromophores

Many squaraines have been observed to exhibit two-photon absorption at transition energies close to those of the lowest energy one-photon electronic transitions. Here, the electronic and vibronic contributions to these low-energy two-photon absorptions are elucidated by performing correlated quantum-chemical calculations on model chromophores that differ in their terminal donor groups (diarylaminothienyl, indolenylidenemethyl, dimethylaminopolyenyl, or 4-(dimethylamino)phenylpolyenyl). For squaraines with diarylaminothienyl and dimethylaminopolyenyl donors and for the longer examples of 4-(dimethylamino)phenylpolyenyl donors, the calculated energies of the lowest two-photon active states approach those of the lowest energy one-photon active (1B(u)) states. This is consistent with the existence of purely electronic channels for low-energy two-photon absorption (TPA) in these types of chromophores. On the other hand, for all squaraines containing indolinylidenemethyl donors, the calculations indicate that there are no low-lying electronic states of appropriate symmetry for TPA. Actually, we find that the lowest energy TPA transitions can be explained through coupling of the one-photon absorption (OPA) active 1B(u) state with b(u) vibrational modes. Through implementation of Herzberg-Teller theory, we are able to identify the vibrational modes responsible for the low-energy TPA peak and to reproduce, at least qualitatively, the experimental TPA spectra of several squaraines of this type.     

De novo design, synthesis, and function of semiartificial myoglobin conjugated with coiled-coil two-alpha-helix peptides

The introduction of a flavin chromophore on the myoglobin (Mb) surface and an effective electron-transfer (ET) reaction through the flavin were successfully achieved by utilizing the self-assembly of heterostranded coiled-coil peptides. We have prepared a semiartificial Mb, named Mb-1alphaK, in which an amphiphilic and cationic alpha-helix peptide is conjugated at the heme propionate (Heme-1alphaK). Heme-1alphaK has a covalently bound iron-protoporphyrin IX (heme) at the N terminus of a 1alphaK peptide sequence. This sequence was designed to form a heterostranded coiled-coil in the presence of a counterpart amphiphilic and anionic 1alphaE peptide sequence in a parallel orientation. Two peptides, Fla(1)-1alphaE and Fla(31)-1alphaE, both incorporating a 10-methylisoalloxazine moiety as an artificial flavin molecule, were also prepared (Fla=2-[7-(10-methyl)isoalloxazinyl]-2-oxoethyl). Heme-1alphaK was successfully inserted into apomyoglobin to give Mb-1alphaK. Mb-1alphaK recognized the flavin-modified peptides and a two-alpha-helix structure was formed. In addition, an efficient ET from reduced nicotinamide adenine dinucleotide to the heme center through the flavin unit was observed. The ET rate was faster in the presence of Fla(1)-1alphaE than in the presence of Fla(31)-1alphaE or the equivalent molecule that has no peptide chain. These results demonstrate that the introduction of a functional chromophore on the Mb surface can be achieved by using specific peptide-peptide interactions. Moreover, the dependence of the ET rate on the position of the flavin indicated that the distance between the heme active site and the flavin chromophore was regulated by the three-dimensional structure of the designed polypeptide.     

Synthesis of chromophores and polyimides with a green chemistry approach for second‐order nonlinear optical applications

This article presents the synthesis of nonlinear optical responsive chromophores by adopting a green chemistry approach by coupling N‐methyl‐N‐(2‐hydroxyethyl)‐4‐amino benzaldehyde with barbituric acid, 1,3‐indanedione, and 1,3‐diethyl‐2‐thiobarbituric acid as the acceptors through stilbene linkage. We performed the synthesis in less than 10 minutes at room temperature with water as a solvent without catalyst. Two different side‐chain polyimides were synthesized from poly(hydroxy‐imide)s with chromophores by Mitsunobu reaction. The chromophores were characterized by Fourier transform infrared, ¹H NMR, ¹³C NMR, and elemental analysis. However, the polyimides were characterized by Fourier transform infrared and ¹H NMR. The inherent viscosities (η_inh) of polyimides were determined by Ubbelohde viscometer, which ranged between 0.1793 and 0.1890 dL/g. The molecular weights of the polyimides were determined using gel permeation chromatography and were in range of 23 000 to 26 000. Polyimides demonstrated an excellent solubility in polar aprotic solvents, indicating good processability. Thermal behavior of these polyimides was studied by differential scanning calorimetry and thermogravimetric analysis. The T_g's were in the range of 185°C to 255°C. The change in the molecular orientation in the polymer films after electrical poling was ascertained using ultraviolet‐visible spectrophotometer and atomic force microscopy. The thicknesses and refractive indices of the thin films were determined by an ellipsometer. The second harmonic generation coefficients of the corona‐poled polymer films at T_opt's, determined by the Maker fringe technique, ranged between 59.33 and 77.82 pm/V. High thermal endurance observed for the polyimides is attributed to the extensive hydrogen bonds in the matrix. The developed polyimides showed no decay in second harmonic generation signals below 110°C, indicating the acceptance for nonlinear optical devices.     

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound‐powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA‐loaded nanomotors to directly penetrate through the plasma membrane of GFP‐expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA‐loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor‐based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.     

A Multi‐Stimuli‐Responsive Oxazine Molecular Switch: A Strategy for the Design of Electrochromic Materials

A multi‐state and multi‐stimuli‐responsive oxazine molecular switch that combines an electro‐base property and sensitive base/acid‐responsive properties was designed and synthesized. The multi‐state structures of the molecular switch, with different colors, were predicted by comparing the optical properties with reference molecules and confirmed by using NMR spectroscopy. The color‐switching mechanism under stimulation with acids and bases was investigated by using DFT calculations. Three single states can be obtained and the switching is unidirectional under acid and base stimulation. The electrochromic phenomenon of the molecular switch, which combines its electro‐base and base‐sensitive properties, was demonstrated. An electrochromic device that exhibited good electrochromic properties with excellent reversibility (2000 cycles) and high coloration efficiency (804 cm² C^?1) was successfully constructed.     

Separation efficiency of free‐solution conjugated electrophoresis with drag‐tags incorporating a synthetic amino acid

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge‐to‐friction ratio in free solution, DNA fragments cannot be separated by size. However, size‐based separation of DNA is possible in free‐solution conjugate electrophoresis (FSCE) if a “drag‐tag” is attached to DNA fragments because the tag breaks the linear charge‐to‐friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free‐solution conjugated electrophoresis, generation of a monodisperse drag‐tag and identification of a strong, site‐specific conjugation method between a DNA fragment and a drag‐tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin‐like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site‐specific conjugation, a methionine residue in drag‐tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.