Toshiaki Furuta

Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein

Green fluorescent protein from the jellyfish (Aequorea GFP) and GFP-like proteins from coral species encode light-absorbing chromophores within their protein sequences. A coral fluorescent protein, Kaede, contains a tripeptide, His(62)-Tyr(63)-Gly(64), which acts as a green chromophore that is photoconverted to red. Here, we present the structural basis for the green-to-red photoconversion. As in Aequorea GFP, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. UV irradiation causes an unconventional cleavage within Kaede protein between the amide nitrogen and the alpha carbon (Calpha) at His(62) via a formal beta-elimination reaction, which requires the whole, intact protein for its catalysis. The subsequent formation of a double bond between His(62)-Calpha and -Cbeta extends the pi-conjugation to the imidazole ring of His(62), creating a new red-emitting chromophore, 2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolinone. The present study not only reveals diversity in the chemical structure of fluorescent proteins but also adds a new dimension to posttranslational modification mechanisms.

Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.

The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell–mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

Real-time analysis of the role of Ca2+ in flagellar movement and motility in single sea urchin sperm

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca2+. We used stroboscopic fluorescence imaging to measure intracellular Ca2+ concentration ([Ca2+]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca2+ entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca2+]i. Instead we propose a new model whereby flagella bending is increased by Ca2+ flux through the nimodipine-sensitive pathway, and is unaffected by [Ca2+]i increases through alternative pathways.

Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6‐bromo‐7‐hydroxycoumarin‐4‐ylmethyl esters of cNMP (Bhc‐cNMP) and their acetyl derivatives (Bhc‐cNMP/Ac) as new caged second messengers. Irradiation of Bhc‐cNMPs quantitatively produced the parent cNMPs with one‐photon uncaging efficiencies (Φε) of up to one order of magnitude better than those of 2‐nitrophenethyl (NPE) cNMPs. In addition, two‐photon induced photochemical release of cNMP from Bhc‐cNMPs (7 and 8) can be observed with the two‐photon uncaging action cross‐sections (δu) of up to 2.28 GM (1 GM=10−50 cm4 s photon−1), which is the largest value among those of the reported Bhc‐caged compounds. The wavelength dependence of the δu values of 7 revealed that the peak wavelength was twice that of the one‐photon absorption maximum. Bhc‐cNMPs showed practically useful water solubility (nearly 500 μM), whereas 7‐acetylated derivatives (Bhc‐cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP‐mediated transduction channels in newt olfactory receptor cells and cAMP‐mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Photoinduced nitric oxide release from a hindered nitrobenzene derivative by two-photon excitation.

Here, we demonstrated photoinduced NO generation from a 2,6-dimethylnitrobenzene-based compound (Flu-DNB) via a two-photon excitation (TPE) process. After pulse laser irradiation to a solution of Flu-DNB, oxidation products of NO were observed. This is the first account of a non-nitrosyl-chelated metal ion containing NO donor which can be controlled by the TPE technique.

New photoremovable protecting groups for carboxylic acids with high photolytic efficiencies at near-UV irradiation. Application to the photocontrolled release of L-glutamate.

We report here the syntheses and the photolytic properties of 3‐(4,5‐dimethoxy‐2‐nitrophenyl)‐2‐butyl (DMNPB) esters as new photoremovable groups for carboxylic acids, and their use for the caging of L‐glutamate. A high‐yielding synthesis of the DMNPB esters led to a 4:1 threo/erythro diastereomeric mixture, which could be separated by HPLC. While these esters were stable in neutral buffer, photolysis at 364 nm induced a ≥95 % release of the carboxylic acid, with a 0.26 quantum yield for L‐glutamate formation. L‐Glutamate release was also possible by two‐photon photolysis with an action cross section of 0.17 GM at 720 nm. Laser photolysis at 350 nm generated a transient species at around 410 nm, attributed to a quinonoid aci‐nitro intermediate that decayed in the submillisecond time range (t1/2=0.53 ms) for the faster γ‐L‐glutamyl threo‐esters. Given the absorbance of these esters (λmax=350 nm; ε=4500), the threo DMNPB esters represent new caging groups that can be efficiently photolyzed at near‐UV wavelengths. An efficient and rapid photolytic release of L‐glutamate has been demonstrated on hippocampal neurons in primary culture.

The E1 Mechanism in Photo-Induced β-Elimination Reactions for Green-to-Red Conversion of Fluorescent Proteins

KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with ultraviolet or violet light. Similar to Kaede and EosFP, two naturally occurring photoconvertible proteins, KikGR contains a His(62)-Tyr(63)-Gly(64) tripeptide sequence, which forms a green chromophore that can be photoconverted to a red one via formal beta-elimination and subsequent extension of a pi-conjugated system. Using a crystallizable variant of KikGR, we determined the structures of both the green and red state at 1.55 A resolution. The double bond between His(62)-C(alpha) and His(62)-C(beta) in the red chromophore is in a cis configuration, indicating that rotation along the His(62) C(alpha)-C(beta) bond occurs following cleavage of the His(62) N(alpha)-C(alpha) bond. This structural rearrangement provides evidence that the beta-elimination reaction governing the green-to-red photoconversion of KikGR follows an E1 (elimination, unimolecular) mechanism.

Photocontrolled Compound Release System Using Caged Antimicrobial Peptide

A novel photocontrolled compound release system using liposomes and a caged antimicrobial peptide was developed. The caged antimicrobial peptide was activated by UV irradiation, resulting in the formation of pores on the liposome surface to release the contained fluorophores. The compound release could be observed using fluorescence measurements and time-lapse fluorescence microscopy. UV irradiation resulted in a quick release of the inclusion compounds (within 1 min in most cases) under simulated physiological conditions. The proposed system is expected to be applicable in a wide range of fields from cell biology to clinical sciences.

Photochemical rearrangement approach to the total synthesis of (±)-pinguisone and (±)-deoxopinguisone

Abstract The total synthesis of (±)-pinguisone and (±)-deoxopinguisone, the unusual [5–6], fused-ring sesquiterpenes, was accomplished by the photochemical transformation of the bicyclo [3.2.2]non-6-en-2-one into the bicyclo [4.3.0]non-4-en-7-one.

Light-activated gene expression from site-specific caged DNA with a biotinylated photolabile protection group

A new method for site-specific caging of plasmid DNA was developed to enable light-activation of gene expression in living cells by exposure to a low dose of light.