Regulation mechanisms of the dual ATPase in KaiC.

KaiC is a dual adenosine triphosphatase (ATPase), with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual-ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to adenosine triphosphate (ATP) is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The up-regulatory effect on the N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB.

Controlled Photodynamic Action of Axial Fluorinated DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin through Self-Aggregation.

DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin (EtP(V)TMPP) and its fluorinated derivative (FEtP(V)TMPP) were synthesized to examine their photodynamic action. These P(V)porphyrins were aggregated in an aqueous solution, resulting in the suppression of their photodynamic activity. In the presence of human serum albumin (HSA), a water-soluble protein, the aggregation states were resolved and formed a binding complex between P(V)porphyrin and HSA. These P(V)porphyrins photosensitized the oxidation of the tryptophan residue of HSA under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was explained by the electron transfer from tryptophan to the photoexcited state of P(V)porphyrins and singlet oxygen generation. The axial fluorination reduced the redox potential of the one-electron reduction of P(V)porphyrin and increased the electron transfer rate constant. However, this axial fluorination decreased the binding constant with HSA, and the quantum yield of photosensitized HSA damage through electron transfer was decreased. The photocytotoxicity of these P(V)porphyrins to HaCaT cells was also confirmed, and FEtP(V)TMPP demonstrated stronger phototoxicity than EtP(V)TMPP. In summary, a self-aggregation of porphyrin photosensitizers and resolving by targeting biomacromolecules may be used to target selective photodynamic action. The redox potential and an association with a targeting biomolecule are the important factors of the electron transfer-mediated mechanism, which is advantageous under hypoxic tumor conditions.

Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria.

The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.

Photosensitized Protein-Damaging Activity, Cytotoxicity, and Antitumor Effects of P(V)porphyrins Using Long-Wavelength Visible Light through Electron Transfer.

Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (1O2) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of 1O2. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.

Multi-frequency ultrasound-assisted cellulase extraction of protein from mulberry leaf: Kinetic, thermodynamic, and structural properties.

The effects of different extraction methods (traditional extraction, ultrasound extraction, cellulase extraction, and ultrasound-assisted cellulase extraction) on the yield of mulberry leaf protein (MLP) were investigated, and the results revealed that multi-frequency ultrasound-assisted cellulase extraction was the most efficient extraction method. The mechanism of the synergistic extraction method used to efficiently extract protein from mulberry leave was investigated, focusing on the kinetics and thermodynamics of the enzymatic process. The results revealed that kinetic parameters KM decreased by 14.07% and kA increased by 5.02%, and the thermodynamic parameters Ea, ΔH, and ΔS decreased by 44.81%, 48.41%, and 21.12 %, respectively, following the process of multi-frequency ultrasound (MFU) pretreatment. The spectral analysis with fluorescence spectra manifested that ultrasound exposed hydrophobic groups and induced molecular unfolding of MLP. Atomic force microscope showed that ultrasound decreased particle size while increasing flexibility of MLP. The effect of ultrasound increases the binding frequency of cellulase and substrates, resulting in greater affinity between the two and promoting the solubilization of MLP. This study provides a theoretical basis to improve the application prospects of MLP.

Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5'-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.

KaiC from a cyanobacterium Gloeocapsa sp. PCC 7428 retains functional and structural properties required as the core of circadian clock system.

KaiC, the core protein of the cyanobacterial clock, assembles into a hexamer upon ATP-binding. The hexameric KaiC from a cyanobacterium Synechococcus elongatus PCC 7942 (Se-KaiC) is a multifunctional enzyme with autokinase, autophosphatase and ATPase and these activities show a circadian rhythm in the presence of two other clock proteins, KaiA and KaiB both in vivo and in vitro. While an interplay among three enzymatic activities has been pointed out through studies on Se-KaiC as the basis of circadian rhythmicity in cyanobacteria, little is known about the structure and functions of KaiC from other cyanobacterial species. In this study, we established a protocol to prepare KaiC from Gloeocapsa sp. PCC 7428 (Gl-KaiC) belonging to a distinct genus from Synechococcus and characterized its oligomeric structure and function. The results demonstrate that Gl-KaiC shares the basic properties with Se-KaiC. The present protocol offers practical means for further analysis of structure and function of Gl-KaiC, which would provide insights into diversity and evolution of the clock systems in cyanobacteria.

Photosensitized enzyme deactivation and protein oxidation by axial-substituted phosphorus(V) tetraphenylporphyrins.

The activity for photodynamic therapy of water-soluble cationic porphyrins, tetraphenylporphyrin P(V) complexes, was investigated. Bis(cyclohexylmethoxy)P(V)tetraphenylporphyrin (DCHMP(V)TPP), dichloroP(V)tetraphenylporphyrin (Cl2P(V)TPP), and dimethoxyP(V)tetraphenylporphyrin (DMP(V)TPP) could cause the photosensitized deactivation of tyrosinase. The tryptophan residue of human serum albumin (HSA) and several kinds of amino acids could be damaged by these P(V)porphyrins under visible light irradiation. The photosensitized damage of these biomolecules was inhibited by sodium azide, a singlet oxygen (1O2) quencher, and enhanced in deuterium oxide, suggesting the contribution of 1O2. However, an excess amount of sodium azide did not completely inhibit the photosensitized damage. In addition, the redox potential measurements demonstrated the possibility of electron transfer from tryptophan and tyrosine to photoexcited P(V)porphyrins. These results suggest that electron transfer-mediated oxidation of amino acids contributes to the photosensitized protein and amino acid damage by these P(V)porphyrins. Specifically, Cl2P(V)TPP showed the highest photodamaging activity in the P(V)porphyrins used in this study. Oxidized products of amino acids by photoexcited P(V)porphyrins were analyzed with a liquid chromatography-mass spectrometer. Because of the hypoxic condition of a tumor, photodynamic therapy through a 1O2-mediated mechanism should be restricted, and the electron transfer-mediated mechanism may improve the photodynamic effect. In the cases of these P(V)porphyrins, redox potential is the most important factor for photosensitized protein and amino acid oxidation through photoinduced electron transfer.