Shared CaM- and S100A1-binding epitopes in the distal TRPM4 N terminus.

The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function.

Cysteine residues mediate high-affinity binding of thioredoxin to ASK1.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1).

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3.

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein.

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry.

Depolarization affects the lateral microdomain structure of yeast plasma membrane.

We report the transmembrane voltage-induced lateral reorganization of highly-ordered lipid microdomains in the plasma membrane of living Saccharomyces cerevisiae. Using trans-parinaric acid (all-trans-9,11,13,15-octadecatetraenoic acid) as a probe of lipid order and different methods of membrane depolarization, we found that depolarization always invokes a significant reduction in the amount of gel-like microdomains in the membrane. Different depolarization mechanisms, including the application of ionophores, cell depolarization by an external electric field, depolarization by proton/hexose co-transport facilitated by HUP1 protein and a reduction of membrane potential caused by compromised respiration efficiency, yielded the same results independently of the yeast strain used.

Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast.

We report sphingolipid-related reorganization of gel-like microdomains in the plasma membrane of living Saccharomyces cerevisiae using trans-Parinaric acid (t-PnA) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Compared to control, the gel-like domains were significantly reduced in the membrane of a sphingolipid-deficient lcb1-100 mutant. The same reduction resulted from sphingolipid depletion by myriocin.

Detailed kinetic analysis of the interaction between the FOXO4-DNA-binding domain and DNA.

The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4-DNA binding domain (FOXO4-DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4-DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4-DBD upon binding to the DNA.

Structural modulation of phosducin by phosphorylation and 14-3-3 protein binding.

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy.

Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction.

Solution structure of the ESCRT-I complex by small-angle X-ray scattering, EPR, and FRET spectroscopy.

ESCRT-I is required for the sorting of integral membrane proteins to the lysosome, or vacuole in yeast, for cytokinesis in animal cells, and for the budding of HIV-1 from human macrophages and T lymphocytes. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the ubiquitin E2 variant and Vps28 C-terminal domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I.

The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms.

Yeast 14-3-3 protein isoforms BMH1 and BMH2 possess a distinctly variant C-terminal tail which differentiates them from the isoforms of higher eukaryotes. Their C-termini are longer and contain a polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which occupies the ligand binding groove and blocks the binding of inappropriate ligands.

14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3).

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit.

Long-term adaptation of Bacillus subtilis 168 to extreme pH affects chemical and physical properties of the cellular membrane.

We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.

Maximum entropy analysis of analytically simulated complex fluorescence decays.

We tested a Maximum Entropy Method developed for oversampled data (SVD-MEM) on complex analytically simulated exponential decay data consisting of both noisy and noiseless multi-exponential fluorescence decay curves. We observed recovery of simulated parameters for three sets of data: a decay containing three exponential functions in both intensity and anisotropy curves, a set of intensity decays composed of 4, 5 and 6 exponential functions, and a decay characterized by a Gaussian lifetime distribution. The SVD-MEM fitting of the noiseless data returned the simulated parameters with the high accuracy.

Glycine-rich loop of mitochondrial processing peptidase alpha-subunit is responsible for substrate recognition by a mechanism analogous to mitochondrial receptor Tom20.

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) alpha-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast alpha-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility.

14-3-3 protein masks the DNA binding interface of forkhead transcription factor FOXO4.

The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal.

Creatine kinase structural changes induced by substrates.

Myofibrillar creatine kinase (CK) buffers the cellular ATP concentration during fluctuating ATP turnover in a muscle. In order to detect structural changes of the CK molecule due to bound substrates, the dynamics of free, ATP-bound, and ATP+creatine-bound CK were examined, using steady-state and time-resolved fluorescence spectroscopy. The intrinsic tryptophan fluorescence of non-labelled CK presented the smaller fluorescence lifetime 2.

Frequency domain fluorometry with pulsed light-emitting diodes.

We present a simple way to extend the time resolution of a standard frequency domain (FD) fluorometer by use of pulsed light-emitting diodes (LEDs) as an excitation source. High temporal resolution of the multifrequency FD method requires the excitation light to be modulated up to the highest possible frequencies with high modulation depth. We used harmonic content of subnanosecond-pulsed LEDs for generation of modulated excitation light.

The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase.

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases.

Temperature modulates binding specificity and affinity of the d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis.

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C.

Monitoring of the proton electrochemical gradient in reconstituted vesicles: quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes.

Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase.

Both the N-terminal loop and wing W2 of the forkhead domain of transcription factor Foxo4 are important for DNA binding.

FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel beta-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors.

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states.

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C.

14-3-3 Protein interacts with nuclear localization sequence of forkhead transcription factor FoxO4.

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS).