Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.

Canonical transient receptor potential (TRPC) channels control influxes of Ca 2؉ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca 2؉ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca 2؉ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca 2؉ -dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.Ca 2ϩ signaling ͉ pyrazole compounds ͉ TRPC channels ͉ TRPC3

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.

Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM). Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed. The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases. A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site. Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared. Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes. When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).

Glutamic acid residues in the SS2 segment of the internal repeats III and IV of the brain calcium channel BI were subjected to single point mutations. The mutant channels were tested for macroscopic current properties and sensitivities to inorganic blockers. The mutation that replaces glutamic acid 1,469 with glutamine altered ion-selection properties and strongly reduced the sensitivity to Cd", whereas the analogous mutation of glutamic acid 1,765 exerted smaller effects on ion-selection properties. Our results indicate that these glutamic acid residues, equivalently positioned in the aligned sequences, play different roles in the selective permeability of the calcium channel.

Point the finger: Zinc‐finger proteins are convenient and site‐selective adaptors for targeting specific locations within DNA‐origami structures. Orthogonal targeting of the specific locations in the structures was demonstrated by using two adaptors, and the application of Escherichia coli lysate that contained the adaptor‐fused proteins successfully afforded the expected protein–DNA assembly.

Arranging quaternary structures by dimerization of monomers is necessary for many sequence-specific DNA-binding proteins to become functional. Upon dimer formation, the DNA contacting regions of each monomer are positioned to a proper orientation that facilitates efficient sequence-specific recognition of DNA.1 Several laboratories have reported model studies that demonstrate the importance of such steric constraints by using covalently bonded dimeric peptides.* 12 However, natural proteins dimerize with noncovalent interactions, and the equilibrium governing the formation of dimers would be important for regulation.3 Recent findings on the transcriptional control mechanisms by heterodimer formation for the basic leucine-zipper protein and the basic helix-loop-helix protein families also indicate that the specificity in dimer formation regulates the sequencespecific DNA binding of such proteins.4 W e report here synthetic oligopeptides that bind to specific DNA sequences upon noncovalent dimer formation. A peptide modified with /3-cyclodextrin (/3-CD) and a peptide with an adamantyl group form a heterodimer mediated by formation of an inclusion complex between the /3-CD and the adamantyl group, and the heterodimer binds to a specific DNA sequence.A 23-residue peptide (G23) derived from the DNA contacting region of the transcriptional activator protein GCN4,5* one of the large family of DNA-binding proteins characterized by a basic leucine-zipper structural motif, was used as a sequencespecific DNA-binding domain. GCN4 binds to the specific DNA sequence upon formation of dimer, which is mediated by a parallelcoiled coil assembly of each leucine-zipper domain.5 Our design for effecting noncovalent dimer formation is a host-guest inclusion complex of /3-CD and its guest compound. Modification of the C-terminal cysteine residue of G23 with mono-6-deoxy-6-iodo-/3-cyclodextrin6 afforded G23-CD, while modification with /V-(bromoacetyl)-1 -adamantanemethylamine gave G23-AD (Figure l).7 It has been shown that 1-adamantaneacetic acid forms a 1:1 inclusion complex in water with /3-cyclodextrin with a dissociation constant of 5 X 10~5 M.8 We have tested whether• To whom correspondence should be addressed.