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Biophysics and Physicobiology

Ryuma Sato, Ryuhei Harada, Yasuteru Shigeta
Photolyases (PHRs) and cryptochromes (CRYs) belong to the same family known as blue-light photoreceptors. Although their amino acid sequences and corresponding structures are similar to each other, they exert different functions. PHRs function as an enzyme to repair UV-induced deoxyribonucleic acid (DNA) lesions such as a cyclobutane pyrimidine dimer (CPD) and a (6-4) photoproduct ((6-4)pp), whereas CRYs are a circadian photoreceptor in plants and animals and at the same time they control the photoperiodic induction of flowering in plants...
2018: Biophysics and Physicobiology
Yoichi Nakatani, Osamu Hisatomi
Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida . Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light-oxygen-voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states...
2018: Biophysics and Physicobiology
Akira Kitamura, Masataka Kinjo
Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis...
2018: Biophysics and Physicobiology
Daisuke Miyashiro, Misato Ohtsuki, Yuta Shimamoto, Jun'ichi Wakayama, Yuki Kunioka, Takakazu Kobayashi, Shin'ichi Ishiwata, Takenori Yamada
We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component...
2017: Biophysics and Physicobiology
Hiroshi Hashimoto, Asami Hishiki, Kodai Hara, Sotaro Kikuchi
DNA damage tolerance (DDT) is a cell function to avoid replication arrest by DNA damage during DNA replication. DDT includes two pathways, translesion DNA synthesis (TLS) and template-switched DNA synthesis (TS). DDT is regulated by ubiquitination of proliferating cell nuclear antigen that binds to double-stranded DNA and functions as scaffold protein for DNA metabolism. TLS is transient DNA synthesis using damaged DNA as a template by error-prone DNA polymerases termed TLS polymerases specialized for DNA damage...
2017: Biophysics and Physicobiology
Hiroyuki Terashima, Akihiro Kawamoto, Yusuke V Morimoto, Katsumi Imada, Tohru Minamino
The bacterial flagellum is a supramolecular motility machine consisting of the basal body as a rotary motor, the hook as a universal joint, and the filament as a helical propeller. Intact structures of the bacterial flagella have been observed for different bacterial species by electron cryotomography and subtomogram averaging. The core structures of the basal body consisting of the C ring, the MS ring, the rod and the protein export apparatus, and their organization are well conserved, but novel and divergent structures have also been visualized to surround the conserved structure of the basal body...
2017: Biophysics and Physicobiology
Kazuho Yoshida, Takahiro Yamashita, Kengo Sasaki, Keiichi Inoue, Yoshinori Shichida, Hideki Kandori
We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template...
2017: Biophysics and Physicobiology
Shota Kondo, Michio Homma, Seiji Kojima
Vibrio alginolyticus normally has a single polar flagellum whose number and placement are regulated positively by FlhF. FlhF is a GTPase and homolog of a signal recognition particle (SRP) protein called Ffh and SRP receptor FtsY. FlhF is located at the cell pole and directs formation of the flagellum. To study the mechanism of FlhF localization, we introduced random mutations into flhF by means of hydroxylamine and isolated mutants that could not generate the flagellum at the cell pole. The novel mutations were only mapped to the GTPase motif of FlhF...
2017: Biophysics and Physicobiology
Naoki Arai, Tadaomi Furuta, Minoru Sakurai
Upon the binding of ATP molecules to nucleotide binding domains (NBDs), ATP-binding cassette (ABC) exporters undergo a conformational transition from an inward-facing (IF) to an outward-facing (OF) state. This molecular event is a typical example of chemo-mechanical coupling. However, the underlying mechanism remains unclear. In this study, we analyzed the IF→OF transition of a representative ABC exporter, MsbA, by solving the equation of motion under an elastic network model (ENM). ATP was represented as a single node in ENM or replaced by external forces...
2017: Biophysics and Physicobiology
Eiji Yamamoto
Many cellular functions, including cell signaling and related events, are regulated by the association of peripheral membrane proteins (PMPs) with biological membranes containing anionic lipids, e.g., phosphatidylinositol phosphate (PIP). This association is often mediated by lipid recognition modules present in many PMPs. Here, I summarize computational and theoretical approaches to investigate the molecular details of the interactions and dynamics of a lipid recognition module, the pleckstrin homology (PH) domain, on biological membranes...
2017: Biophysics and Physicobiology
Toshifumi Kumai
It is established knowledge that the action potential event of nerves is formed by the combination of a phasic inward Na(+) current and a following outward K(+) current which increases gradually. These changes in current are commonly referred to as conductance changes of channels for Na(+) and K(+) with time. On the other hand, electric requirements for action potential generation in phenomena such as anode break excitation, hyperpolarizing break stimulation and accommodation, strongly suggest an existence of an inductance factor in the plasma membrane of nerves...
2017: Biophysics and Physicobiology
Ayumi Hashimoto, Toyohiro Sawada, Kiyohisa Natsume
The study aimed to determine whether and how the activation of the acetylcholine receptor affects epileptiform discharges in the CA3 region in a rat hippocampus. Picrotoxin (100 μM), a GABAA receptor antagonist, was applied to a hippocampal slice to induce epileptiform discharges. The effects of the cholinergic agonist, carbachol, on the discharges were examined at the several concentrations (1-30 μM). Carbachol had different impacts on epileptiform discharges at the different concentrations. Relatively low concentrations of carbachol (<10 μM) increased the frequency but decreased the amplitude of the discharges...
2017: Biophysics and Physicobiology
Mikihiro Shibata, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, Ryohei Yasuda
Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell...
2017: Biophysics and Physicobiology
Hiroaki Machiyama, Takamitsu J Morikawa, Kazuko Okamoto, Tomonobu M Watanabe, Hideaki Fujita
We evaluated usability of a previously developed genetically encoded molecular crowding sensor in various biological phenomena. Molecular crowding refers to intracellular regions that are occupied more by proteins and nucleotides than by water molecules and is thought to have a strong effect on protein function. To evaluate intracellular molecular crowding, usually the diffusion coefficient of a probe is used because it is related to mobility of the surrounding molecular crowding agents. Recently, genetically encoded molecular crowding sensors based on Förster resonance energy transfer were reported...
2017: Biophysics and Physicobiology
Yutaro Yamada, Hiroki Konno, Katsuya Shimabukuro
In this study, we present a new technique called correlative atomic force and transmission electron microscopy (correlative AFM/TEM) in which a targeted region of a sample can be observed under AFM and TEM. The ultimate goal of developing this new technique is to provide a technical platform to expand the fields of AFM application to complex biological systems such as cell extracts. Recent advances in the time resolution of AFM have enabled detailed observation of the dynamic nature of biomolecules. However, specifying molecular species, by AFM alone, remains a challenge...
2017: Biophysics and Physicobiology
Akira R Kinjo
A grand canonical Monte Carlo (MC) algorithm is presented for studying the lattice gas model (LGM) of multiple protein sequence alignment, which coherently combines long-range interactions and variable-length insertions. MC simulations are used for both parameter optimization of the model and production runs to explore the sequence subspace around a given protein family. In this Note, I describe the details of the MC algorithm as well as some preliminary results of MC simulations with various temperatures and chemical potentials, and compare them with the mean-field approximation...
2017: Biophysics and Physicobiology
Kotomi Shibata, Tsubasa Koyama, Shohei Inde, Sosuke Iwai, Shigeru Chaen
The myosin II SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain and possibly plays a key role in the arrangement of the converter/lever arm. Several point mutations within the SH1 helix in human myosin IIs have been shown to cause diseases. To reveal whether these SH1 helix mutations affect not only motile activities but also thermal properties of myosin II, here we introduced the E683K or R686C point mutation into the SH1 helix in Dictyostelium myosin II. Thermal inactivation as well as thermal aggregation rates of these mutant proteins demonstrated that these mutations decreased the thermal stability of myosin II...
2017: Biophysics and Physicobiology
Yumeka Yamauchi, Masae Konno, Shota Ito, Satoshi P Tsunoda, Keiichi Inoue, Hideki Kandori
Microbial rhodopsins are membrane proteins found widely in archaea, eubacteria and eukaryotes (fungal and algal species). They have various functions, such as light-driven ion pumps, light-gated ion channels, light sensors and light-activated enzymes. A light-driven proton pump bacteriorhodopsin (BR) contains a DTD motif at positions 85, 89, and 96, which is unique to archaeal proton pumps. Recently, channelrhodopsins (ChRs) containing the DTD motif, whose sequential identity is ~20% similar to BR and to cation ChRs in Chlamydomonas reinhardtii (CrCCRs), were found...
2017: Biophysics and Physicobiology
Jun Tamogami, Takashi Kikukawa, Toshifumi Nara, Makoto Demura, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama, Seiji Miyauchi, Kazumi Shimono, Naoki Kamo
A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M⇄N⇄O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→)...
2017: Biophysics and Physicobiology
Noriyo Mitome, Hiroki Sato, Taishi Tomiyama, Katsuya Shimabukuro, Takuya Matsunishi, Kohei Hamada, Toshiharu Suzuki
The Fo-a subunit of the Na(+)-transporting FoF1 ATP synthase from Propionigenium modestum plays a key role in Na(+) transport. It forms half channels that allow Na(+) to enter and leave the buried carboxyl group on Fo-c subunits. The essential Arg residue R226, which faces the carboxyl group of Fo-c subunits in the middle of transmembrane helix 5 of the Fo-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid FoF1 (Fo from P...
2017: Biophysics and Physicobiology
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