Modulation Domain Research Articles

Modulated phase ofK2MoxW1−xO4mixed compounds using orthorhombic symmetry: A powder diffraction study

High-resolution x-ray powder diffraction data have been used to study the modulated phase of the ${\text{K}}_{2}{\text{WO}}_{4}$, ${\text{K}}_{2}{\text{Mo}}_{0.5}{\text{W}}_{0.5}{\text{O}}_{4}$, and ${\text{K}}_{2}{\text{MoO}}_{4}$ compounds. Literature considers ${\text{K}}_{2}{\text{WO}}_{4}$ and ${\text{K}}_{2}{\text{MoO}}_{4}$ as exceptions among the members of ${A}^{\ensuremath{'}}{A}^{\ensuremath{''}}B{X}_{4}$ family because their thermotropic phase transitions do not follow the commonly observed sequence and because their modulation wave vector, unusually, lies on the pseudohexagonal plane. Until now, the modulated phases of these compounds have been interpreted using hexagonal symmetry. The present work indicates, however, that a model consisting of threefold twinned orthorhombic modulated domains better describes the satellites observed in the temperature range of 620--740 K. The $Ccmm$ space group is proposed for the average structure of the whole family ${\text{K}}_{2}{\text{Mo}}_{x}{\text{W}}_{1\ensuremath{-}x}{\text{O}}_{4}$, whereas the $Ccmm(\ensuremath{\alpha}00)000$ with the wave vector $\mathbit{q}=(0.5+\ensuremath{\delta}){\mathbit{a}}^{\ensuremath{\ast}}$ is the appropriate superspace group for the description of the modulated structures. The coexistence of three orthorhombic domains, each of which is rotated by $120\ifmmode^\circ\else\textdegree\fi{}$ from each other, indeed reproduces a pseudohexagonal basis, which is a common feature in the ${A}^{\ensuremath{'}}{A}^{\ensuremath{''}}B{X}_{4}$ family. The usage of the twinning model allows a much simpler interpretation of the diffraction pattern and provides a more consistent way to describe the phase-transition sequence shown by the mixed ${\text{K}}_{2}{\text{Mo}}_{x}{\text{W}}_{1\ensuremath{-}x}{\text{O}}_{4}$ crystals.

Hysteresis suppression and synchronization near 3:1 subharmonic resonance

Effect of a high-frequency excitation on hysteresis and synchronization area in a forced van der Pol–Duffing oscillator near the subharmonic 3:1 resonance is analyzed. By means of perturbation techniques, the 3:1 resonance area and the quasiperiodic modulation domain are captured. The results shown that adding a high-frequency excitation causes the backbone curves to shift and to bend from softening to hardening. The results also reveal that a high-frequency excitation significantly affects the resonance and the synchronization zones. Further, jump phenomenon occurring in a certain range of the forcing frequency suggesting that hysteresis near the 3:1 subharmonic resonance cannot be ignored in practical applications.

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels

In immune cells, generation of sustained Ca(2+) levels is mediated by the Ca(2+) release-activated Ca(2+) (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca(2+) sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca(2+) stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233-450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50-74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca(2+) currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233-420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233-485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.

The concept of signal-to-noise ratio in the modulation domain and speech intelligibility

A new concept is proposed that relates to intelligibility of speech in noise. The concept combines traditional estimations of signal-to-noise ratios (S/N) with elements from the modulation transfer function model, which results in the definition of the signal-to-noise ratio in the modulation domain: the (SN)(mod). It is argued that this (SN)(mod), quantifying the strength of speech modulations relative to a floor of spurious modulations arising from the speech-noise interaction, is the key factor in relation to speech intelligibility. It is shown that, by using a specific test signal, the strength of these spurious modulations can be measured, allowing an estimation of the (SN)(mod) for various conditions of additive noise, noise suppression, and amplitude compression. By relating these results to intelligibility data for these same conditions, the relevance of the (SN)(mod) as the key factor underlying speech intelligibility is clearly illustrated. For instance, it is shown that the commonly observed limited effect of noise suppression on speech intelligibility is correctly "predicted" by the (SN)(mod), whereas traditional measures such as the speech transmission index, considering only the changes in the speech modulations, fall short in this respect. It is argued that (SN)(mod) may provide a relevant tool in the design of successful noise-suppression systems.

Windowless Open Photoacoustic Helmholtz Cell

A phenomenon which is usually considered as the main disadvantage of photoacoustic Helmholtz cells is photoacoustic background signal resulting from the light absorption by the cell windows. Although there are methods which reduce this background signal, e.g. light wavelength modulation or time domain flltering of the photoacoustic response from the sample, the methods are limited to particular applications, and none of them would completely remove the mentioned background signal. The paper presents design of a windowless open photoacoustic Helmholtz cell, and preliminary results of frequency response measurements of such a cell. Removal of the windows eliminated the main source of the photoacoustic background signal. Additionally, the proposed design allows for unrestricted or forced ∞ow of the ∞uid fllling the cell. As a result, the cell can be easily adapted for continuous, real-time photoacoustic measurements. Under proper selection of the cell dimensions, Q-factors of such open photoacoustic Helmholtz resonators are comparable to the Q-factors of corresponding closed Helmholtz cells.

A High-Precision Time Interval Measurement Method Using Phase-Estimation Algorithm

High-precision time interval measurement is widely used in time synchronization, satellite navigation, laser ranging, and nuclear electronics. The resolution and accuracy of the currently used time counter are about 25 and 100 ps, respectively. A new time interval measurement method, in which the signal under test is used to trigger a sampler and the phase of the reference sinusoid signal is sampled to record the time stamp of the test signal, was put forward. Then, the sampled reference sine signal's phase is estimated by the interpolated fast Fourier transform (FFT), and 1-ps resolution and accuracy can theoretically be achieved. The test experiment of the prototype instrument shows that a 10-ps accuracy has been reached. If the hardware prototype is improved to reduce trigger jitter and sampling clock jitter, the measurement instrument may reach 1-ps accuracy. The measurement system is used not only for time interval measurement but also for period measurement and even modulation domain analysis.

Hysteresis suppression for primary and subharmonic 3:1 resonances using fast excitation

We analyze the effect of a fast harmonic excitation on hysteresis and on entrainment area in a forced van der Pol–Duffing oscillator near the primary and the 3:1 subharmonic resonances. Analytical treatment based on perturbation techniques is performed to capture the entrainment zone, the quasiperiodic modulation domain and the hysteresis area in the vicinity of the two resonances. Specifically, it is shown that a fast harmonic excitation can suppress hysteresis for a certain range of the fast excitation leading to a smooth transition between the quasiperiodic and the frequency-locked responses near these resonances. Furthermore, the influence of different system parameters on the hysteresis area has been investigated. In particular, the results reveal that the amplitude of the fast excitation and the nonlinear damping significantly affect the domain of hysteresis suppression near the primary and the 3:1 subharmonic resonances.

Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones.

BackgroundMycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001.ResultsWe report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone FConclusionThe mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.

Effects of spectral modulation filtering on vowel identification

The goal of this study was to measure the effects of global spectral manipulations on vowel identification by progressively high-pass filtering vowel stimuli in the spectral modulation domain. Twelve American-English vowels, naturally spoken by a female talker, were subjected to varied degrees of high-pass filtering in the spectral modulation domain, with cutoff frequencies of 0.0, 0.5, 1.0, 1.5, and 2.0 cycles/octave. Identification performance for vowels presented at 70 dB sound pressure level with and without spectral modulation filtering was measured for five normal-hearing listeners. Results indicated that vowel identification performance was progressively degraded as the spectral modulation cutoff frequency increased. Degradation of vowel identification was greater for back vowels than for front or central vowels. Detailed acoustic analyses indicated that spectral modulation filtering resulted in a more crowded vowel space (F1xF2), reduced spectral contrast, and reduced spectral tilt relative to the original unfiltered vowels. Changes in the global spectral features produced by spectral modulation filtering were associated with substantial reduction in vowel identification. The results indicated that the spectral cues critical for vowel identification were represented by spectral modulation frequencies below 2 cycles/octave. These results are considered in terms of the interactions among spectral shape perception, spectral smearing, and speech perception.

Stereospecificity of Ketoreductase Domains 1 and 2 of the Tylactone Modular Polyketide Synthase

Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.

Novel Intermolecular Iterative Mechanism for Biosynthesis of Mycoketide Catalyzed by a Bimodular Polyketide Synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-β-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel “modularly iterative” mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.

Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the d form of the amino acid residues while the two following modules recognize the l form and the last module the l or d form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export.

In vivo Mutational Analysis of the Mupirocin Gene Cluster Reveals Labile Points in the Biosynthetic Pathway: the “Leaky Hosepipe” Mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.

Neurocalcin δ Modulation of ROS-GC1, a New Model of Ca2+ Signaling

ROS-GC1 membrane guanylate cyclase is a Ca(2+) bimodal signal transduction switch. It is turned "off" by a rise in free Ca(2+) from nanomolar to the semicromolar range in the photoreceptor outer segments and the olfactory bulb neurons; by a similar rise in the bipolar and ganglion retinal neurons it is turned "on". These opposite operational modes of the switch are specified by its Ca(2+) sensing devices, respectively termed GCAPs and CD-GCAPs. Neurocalcin delta is a CD-GCAP. In the present study, the neurocalcin delta-modulated site, V(837)-L(858), in ROS-GC1 has been mapped. The location and properties of this site are unique. It resides within the core domain of the catalytic module and does not require the alpha-helical dimerization domain structural element (amino acids 767-811) for activating the catalytic module. Contrary to the current beliefs, the catalytic module is intrinsically active; it is directly regulated by the neurocalcin delta-modulated Ca(2+) signal and is dimeric in nature. A fold recognition based model of the catalytic domain of ROS-GC1 was built, and neurocalcin delta docking simulations were carried out to define the three-dimensional features of the interacting domains of the two molecules. These findings define a new transduction model for the Ca(2+) signaling of ROS-GC1.

Improvement of speech recognition thresholds by spectral modulation enhancement

Our previous studies have shown that vowel identification in noise was significantly improved by spectral enhancement resulting from modifications in the spectral modulation domain. The present study investigates whether spectral enhancement results in improved speech recognition in background competition. Speech recognition thresholds (SRT) were measured in a long-term speech shaped (LTSS) noise and in multi-talker babble using NU-6 words with and without spectral enhancement for young normal-hearing listeners. Results indicated that SRTs were significantly reduced, by 2.2 dB, in LTSS noise and in babble when speech sounds were spectrally enhanced between 2.0 and 2.5 cycles/octave, while no significant changes in SRTs occurred for enhancement between 1.5 and 2 cycles/octave or between 1.5 and 2.5 cycles/octave. Spectral modulation transfer functions were also measured for each listener. Linear regression analyses showed that SRT improvement was moderately correlated with modulation detection thresholds at a spectral modulation frequency of 0.5 cycles/octave. Thus, it appears that better modulation detection at low spectral modulation frequencies is associated with greater improvement in word recognition in background competition.

Suppression of hysteresis in a forced van der Pol–Duffing oscillator

This paper examines the suppression of hysteresis in a forced nonlinear self-sustained oscillator near the fundamental resonance. The suppression is studied by applying a rapid forcing on the oscillator. Analytical treatment based on perturbation analysis is performed to capture the entrainment zone, the quasiperiodic modulation domain and the hysteresis area as well. The analysis leads to a strategy for the suppression of hysteresis occurring between 1:1 frequency-locked motion and quasiperiodic response. This hysteresis suppression causes the disappearance of nonlinear effects leading to a smooth transition between the quasiperiodic and the frequency-locked responses. Specifically, it appears that a rapid forcing introduces additional apparent nonlinear stiffness which can effectively suppress hysteresis in a certain range of the rapid excitation frequency. This work was motivated by the important issue of controlling and eliminating hysteresis often undesirable in mechanical systems, in general, and in application to microscale devices, especially.

Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.

Synthesis of Tumor Necrosis Factor Alpha-Induced Protein 6 in Porcine Preovulatory Follicles: A Study with A38 Antibody1

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.

Roles of Functional and Structural Domains of Hepatocyte Growth Factor Activator Inhibitor Type 1 in the Inhibition of Matriptase

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.

A Point Mutation in atpC1 Raises the Redox Potential of the Arabidopsis Chloroplast ATP Synthase γ-Subunit Regulatory Disulfide above the Range of Thioredoxin Modulation

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the gamma-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the gamma-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.