Shigella sonnei utilises colicins during inter-bacterial competition.

The mammalian colon is one of the most densely populated habitats currently recognised, with 1011-1013 commensal bacteria per gram of colonic contents. Enteric pathogens must compete with the resident intestinal microbiota to cause infection. Among these enteric pathogens are Shigella species which cause approximately 125 million infections annually, of which over 90 % are caused by Shigella flexneri and Shigella sonnei. Shigella sonnei was previously reported to use a Type VI Secretion System (T6SS) to outcompete E. coli and S. flexneri in in vitro and in vivo experiments. S. sonnei strains have also been reported to harbour colicinogenic plasmids, which are an alternative anti-bacterial mechanism that could provide a competitive advantage against the intestinal microbiota. We sought to determine the contribution of both T6SS and colicins to the anti-bacterial killing activity of S. sonnei. We reveal that whilst the T6SS operon is present in S. sonnei, there is evidence of functional degradation of the system through SNPs, indels and IS within key components of the system. We created strains with synthetically inducible T6SS operons but were still unable to demonstrate anti-bacterial activity of the T6SS. We demonstrate that the anti-bacterial activity observed in our in vitro assays was due to colicin activity. We show that S. sonnei no longer displayed anti-bacterial activity against bacteria that were resistant to colicins, and removal of the colicin plasmid from S. sonnei abrogated anti-bacterial activity of S. sonnei. We propose that the anti-bacterial activity demonstrated by colicins may be sufficient for niche competition by S. sonnei within the gastrointestinal environment.

Associations between diet composition, dietary pattern, and weight outcomes after bariatric surgery: a systematic review.

INTRODUCTION: Literature describing the impact of dietary intake on weight outcomes after bariatric surgery has not been synthesized. This study aimed to synthesize the evidence regarding any association between diet composition and weight outcomes post-bariatric surgery. METHODS: CINAHL, Cochrane, Embase, MEDLINE and Scopus were searched for adult studies up to June 2021 that assessed any association between dietary intakes (≥1-macronutrient, food group, or dietary pattern) and weight outcomes at 12-months or longer after bariatric surgery. Risk of bias and quality assessments were conducted using the Scottish Intercollegiate Guidelines Network checklists and the NHMRC's Level of Evidence and Grades for Recommendations. Study findings were presented according to the time of post-surgery dietary intake assessment (≤12months, between 12 and 24 months, ≥24months). RESULTS: 5923 articles were identified, 260 were retrieved for full text screening, and 36 were eligible for inclusion (9 interventional including five randomized-controlled trials, and 27 observational cohort studies; sample sizes: 20-1610; total sample: 5065; follow-up periods: 1 year-12 years; level of evidence: II to IV, risk of bias: low to high). Findings on the association between long-term weight outcomes and dietary composition up to 24-months were mixed. After 24-months, studies consistently suggested no significant associations between weight loss and macronutrient composition or core food group patterns, or between carbohydrate, protein or food group patterns and weight recurrence. A single cohort study reported a weak association between diet quality score and weight-recurrence after 24-months. CONCLUSION: There was no strong evidence to support significant associations between diet composition and weight outcomes post-bariatric surgery. The heterogeneity in study design and quality may reduce generalizability to external populations. Individualized dietary recommendations may be useful to support long-term post-surgery weight outcomes. More studies are needed to define and measure diet quality in this patient cohort. REGISTRATION: PROSPERO (CRD42021264120).

Association between biochemical control and comorbidities in patients with acromegaly: an Italian longitudinal retrospective chart review study.

PURPOSE: Achieving biochemical control (normalization of insulin-like growth factor-1 [IGF-1] and growth hormone [GH]) is a key goal in acromegaly management. However, IGF-1 and GH fluctuate over time. The true potential impact of time-varying biochemical control status on comorbidities is unclear and relies on multiple, longitudinal IGF-1 and GH measurements. This study assessed the association between time-varying biochemical control status and onset of selected comorbidities in patients with acromegaly. METHODS: Medical charts of adults with confirmed acromegaly and ≥ 6 months of follow-up at an Italian endocrinology center were reviewed. Patients were followed from the first diagnosis of acromegaly at the center until loss to follow-up, chart abstraction, or death. Biochemical control status was assessed annually and defined as IGF-1 ≤ the upper limit of normal, or GH ≤ 2.5 µg/L in the few cases where IGF-1 was unavailable. Time-varying Cox models were used to assess the association between biochemical control status and comorbidities. RESULTS: Among 150 patients, 47% were female, average age at diagnosis was 43.1, and mean length of follow-up was 10.4 years. Biochemical control was significantly associated with a lower hazard of diabetes (HR = 0.36, 95% CI 0.15; 0.83) and cardiovascular system disorders (HR = 0.54, 95% CI 0.31; 0.93), and a higher hazard of certain types of arthropathy (HR = 1.68, 95% CI 1.04; 2.71); associations for other comorbidities did not reach statistical significance. CONCLUSION: Results further support the importance of achieving biochemical control, as this may reduce the risk of high-burden conditions, including diabetes and cardiovascular system disorders. The association for arthropathy suggests irreversibility of this impairment. Due to limitations, caution is required when interpreting these results.

Diagnostic accuracy of spot urine protein-to-creatinine ratio for proteinuria and its association with adverse pregnancy outcomes in Chinese pregnant patients with pre-eclampsia.

INTRODUCTION: International guidelines have endorsed spot urine protein-to-creatinine ratio of >30 mg protein/mmol creatinine as an alternative to a 24-hour urine sample to represent significant proteinuria. This study aimed to determine the accuracy of spot urine protein-to-creatinine ratio in predicting significant proteinuria and adverse pregnancy outcome. METHODS: This case series was conducted in a regional obstetric unit in Hong Kong. A total of 120 Chinese pregnant patients with pre-eclampsia delivered at Queen Elizabeth Hospital from January 2011 to December 2013 were included. Relationship of spot urine protein-to-creatinine ratio and 24-hour proteinuria; accuracy of the ratio against 24-hour urine protein at different cut-offs; and relationship of such ratio and adverse pregnancy outcome were studied. RESULTS: Spot urine protein-to-creatinine ratio was correlated with 24-hour urine protein with Pearson correlation coefficient of 0.914 (P<0.0001) when the ratio was <200 mg/mmol. The optimal threshold of spot urine protein-to-creatinine ratio for diagnosing proteinuria in Chinese pregnant patients (33 mg/mmol) was similar to that stated in the international literature (30 mg/mmol). A cut-off of 20 mg/mmol provided a 100% sensitivity, and 52 mg/mmol provided a 100% specificity. There was no significant difference in spot urine protein-to-creatinine ratio between cases with and without adverse pregnancy outcome. CONCLUSIONS: Spot urine protein-to-creatinine ratio had a positive and significant correlation with 24-hour urine results in Chinese pre-eclamptic women when the ratio was <200 mg/mmol. Nonetheless, this ratio was not predictive of adverse pregnancy outcome.

Development of miconazole nitrate containing chitosan microcapsules and their anti-Aspergillus niger activity.

In this article, we report the development of chitosan/miconazole nitrate microcapsules. Four miconazole nitrate ratios including 12.5, 25, 50 and 100 mg were performed in the chitosan-based microencapsulation system. Chitosan microcapsules with the drug input of 25 mg showed the highest encapsulation efficiency (52.47%) and acceptable mean particle size (5.65 µm) when compared with those of 12.5, 50 and 100 mg. Fourier transform infrared spectroscopic spectrum proved the entrapment of miconazole nitrate into chitosan microcapsules. The antifungal result demonstrated that microcapsules containing 75 µg miconazole nitrate possessed comparable anti-Aspergillus niger activity as the commercial ointment. The growth inhibition of miconazole nitrate containing chitosan microcapsules towards human skin keratinocytes was found to be dose dependent. A total of 75 µg of miconazole nitrate containing microcapsules revealed about 25% of growth inhibition while that of 150 µg showed approximately 70% of growth inhibition. Special monitoring should be taken if a higher dose of miconazole nitrate was used to develop the microcapsules.

Actin filaments in sensory hairs of inner ear receptor cells.

Receptor cells in the ear are excited through the bending of sensory hairs which project in a bundle from their surface. The individual stereocilia of a bundle contain filaments about 5 nm in diameter. The identity of these filaments has been investigated in the crista ampullaris of the frog and guinea pig by a technique of decoration with subfragment-1 of myosin (S-1). After demembranation with Triton X-100 and incubation with S-1, "arrowhead" formation was observed along the filaments of the stereocilia and their rootlets and also along filaments in the cuticular plate inside the receptor cell. The distance between attached S-1 was 35 nm and arrowheads pointed in towards the cell soma. It is concluded that the filaments of stereocilia are composed of actin.

JEM spotlight: Environmental monitoring of airborne nanoparticles.

The aim of this work was to review the existing instrumental methods to monitor airborne nanoparticles in different types of indoor and outdoor environments in order to detect their presence and to characterise their properties. Firstly the terminology and definitions used in this field are discussed, which is followed by a review of the methods to measure particle physical characteristics including number, concentration, size distribution and surface area. An extensive discussion is provided on the direct methods for particle elemental composition measurements, as well as on indirect methods providing information on particle volatility and solubility, and thus in turn on volatile and semivolatile compounds of which the particle is composed. A brief summary of broader considerations related to nanoparticle monitoring in different environments concludes the paper.

Seasonal variation of chemical characteristics of fine particulate matter at a high-elevation subtropical forest in East Asia.

The aim of this study was to chemically characterize the fine particulate matter (PM2.5) at a subtropical forest in East Asia under the influences of anthropogenic and biogenic sources and a complex topographic setting. Four seasonal campaigns were conducted at the Xitou Experimental Forest in central Taiwan from the winter of 2013 to the autumn of 2014. The results indicated that the ambient levels and chemical features of PM2.5 exhibited pronounced seasonal variations. Non-sea-salt sulfate (nss-SO42-) constituted the major component of PM2.5, followed by ammonium (NH4+) and nitrate (NO3-) during winter, summer and autumn. However, it was revealed that the mass fraction of NO3- increased to be comparable with that of nss-SO42- in springtime. The mass contribution of secondary organic carbon (SOC) to PM2.5 peaked in summer (13.2%), inferring the importance of enhanced photo-oxidation reactions in SOC formation. Diurnal variations of O3 and SO2 coincided with each other, suggesting the transport of aged pollutants from distant sources, whereas CO and NOx were shown to be under the influences of both local and regional sources. Notably high sulfur oxidation ratio (SOR) and nitrogen oxidation ratio (NOR) were observed, which were 0.93 ±â€¯0.05 and 0.39 ±â€¯0.20, respectively. Precursor gases (i.e. SO2 and NOx) could be converted to sulfate and nitrate during the transport by the uphill winds. Furthermore, due to the high relative humidity at Xitou, enhanced aqueous-phase and/or heterogeneous reactions could further contribute to the formation of sulfate and nitrate at the site. This study demonstrated the significant transport of urban pollutants to a subtropical forest by the mountain-valley circulations as well as the long-range transport from regional sources, whereas the implications of which for regional climate change necessitated further investigation.

Mechanisms of intranephronal proteinaceous cast formation by low molecular weight proteins.

Proteinaceous cast formation in the distal nephron of the kidney from low molecular weight proteinuria is a significant, but poorly characterized, cause of renal failure. To study this phenomenon, we: (a) microperfused the loop segment (LS) of rats in vivo with artificial tubule fluid (ATF) containing four different low molecular weight proteins, 0.01-50 mg/ml, to detect alterations in LS function, and (b) examined the interaction between several proteins and Tamm-Horsfall glycoprotein (THP) in vitro with turbidity and dynamic light-scattering measurements. Perfusion of the LS for less than 2 min with cast-forming proteins (Bence Jones protein [BJP3] and myoglobin) decreased chloride absorption and elevated early distal tubule fluid (ED) [Cl-], compared with results obtained with control perfusions that used ATF alone. BJP3 decreased chloride absorption in a concentration-dependent fashion. Perfusion with non-cast-forming proteins (albumin and BJP1) enhanced chloride absorption and decreased ED [Cl-]. In vitro, proteins that had isoelectric points (pI) greater than 5.1 aggregated with THP. Aggregation was enhanced with increasing [NaCl] or [CaCl2]. Albumin (pI 4.8) and beta-lactoglobulin (pI 5.1) did not coprecipitate. The molecular size of THP alone increased when [NaCl] greater than 80 mM. Thus, cast-forming proteins aggregated with THP in vitro and caused in vivo LS dysfunction, which elevated ED [Cl-], facilitating aggregation. In contrast, non-cast-forming proteins either did not interact with THP or lowered ED [Cl-], which did not provide an environment for aggregation. Altered LS function and interaction of some proteins with THP were related to different physicochemical properties of the proteins and independently contributed to the formation of proteinaceous casts in the kidney.

Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84.

Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain an insight about the nature of Ca(2+)-induced conformational changes in the N-domain regulatory region of cTnC. Changes in the experimental emission spectra, quantum yields, and excited-state lifetimes suggested that bound Ca2+ at the single regulatory site induced a less polar microenvironment for both probes attached to Cys-84. However, a twofold increase in the bimolecular collisional quenching constant was observed for both probes in the presence of activator Ca2+, indicating an increased exposure of the probes to solvent. These data were interpreted with reference to the origins of the observed Stokes' shifts. In the apo and 2Mg states of cTnC, the attached probes were partially shielded by helices B and C, and their excited-states were highly quenched in the tertiary structure through strong interactions of a dipolar nature with neighboring amino-acid side chains. In the 3Ca state, these interactions were disrupted so that nonradiative decay processes were suppressed and radiative processes were enhanced, leading to the observed increases in quantum yields and lifetimes and blue-shifts of the emission spectra. As the disruption of internal quenching resulted from separation of helices B and C from helix D, the attached probes became more accessible to solvent and experienced increases in the rate of collisions with external molecules in the solvent. Although this increased exposure to solvent would lead to suppression of radiative decay processes, this effect apparently was overcompensated by the effect of elimination of internal quenching. The present results are consistent with a Ca(2+)-induced open conformation of the N-domain in cTnC.

Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.

An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry.

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by Förster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl] amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C.

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.

Proximity relationship in the binary complex formed between troponin I and troponin C.

We have determined six molecular distances among four sites in the binary complex formed between troponin C (TnC) and troponin I (TnI) by fluorescence resonance energy transfer between donor and acceptor probes that were either an intrinsic fluorophore (Trp158 of TnI) or extrinsic probes attached to the sites. The three extrinsic probes were dansylaziridine (DNZ), N'-(iodoacetyl)-N'-(8-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 5-(iodoacetamido)eosin (IAE). The four fluorophores provided four donor-acceptor pairs: DNZ----IAE, Trp----IAEDANS, IAEDANS----IAE, and Trp----DNZ. They allowed determinations of separations between specific sites from measurements of energy transfer from (1) Met25 (DNZ) to Cys98 (IAE) in TnC, (2) Trp158 to Cys133 (IAEDANS) in TnI, (3) Cys98 (IAEDANS) of TnC to Cys133(IAE) of TnI, (4) Trp158 of TnI to Cys98(IAEDANS) of TnC, and (6) Met25(DNZ) of TnC to Cys133(IAE) of TnI. Distance (1) in TnC was little affected when the isolated protein was complexed with TnI, whereas distance (2) in TnI increased by 6A (29%) when TnI was incorporated into the binary complex. In the presence of EGTA, the six donor-acceptor separations (R) in the complex were in the range 28 to 57 A based on kappa 2 = 2/3. Mg2+ had only small effects on R, but Ca2+ induced substantial increases or decreases of R in five of the six distances. These changes were not accompanied by significant changes in the axial depolarization of the fluorophores. The results indicate global structural perturbations of regions of the two proteins in the complex by Ca2+ binding to the TnC, and suggest that large-scale movements of domains of troponin subunits may be the initial molecular events that occur in the transmission of the Ca2+ signal in the regulation of contraction by calcium.

Tropomyosin length and two-stranded F-actin flexibility in the thin filament.

In muscle thin filaments, each tropomyosin molecule is considered to be a rope-like structure that winds along the filament in contact with seven consecutive actin monomers on the same strand of the two-stranded actin helix. Taking into account the head-to-tail overlap of the tropomyosin molecule, the effective length of this "rope" is about 405 angstrum, which is believed to be conserved. Tropomyosin appears to be neither extensible nor compressible in its axial direction, although it may possess much flexibility in the transverse direction. During the "maximally on" state, characterized by the presence of Ca2+ and the strong binding between actin and myosin subfragment 1, the following conditions are thought to occur: the motion and associated flexibility of tropomyosin are reduced; the actin filament flexibility increases; a maximum number of equivalent tropomyosin binding sites on actin are concurrently saturated; and the tropomyosin molecule maintains an average thin filament radius of 38 to 40 angstrum. Under these potentiated conditions, the length of tropomyosin can be used to determine the limits on the underlying "cumulative angular disorder" of the actin filament with which it interacts. Our calculations show that only a small amount (approximately 1 to 3 degrees) of this type of actin monomer rotational disorder is possible at this stage of the contractile cycle, unless the length of the tropomyosin molecule is increased substantially between the head-to-tail joints. However, if the dominant type of F-actin rotational flexibility is between two relatively rigid actin strands (the lateral slipping/rotational offset model), all of the above actin-tropomyosin interactions can be completely and easily accommodated. We also discuss the implications of an interdomain hinge in G-actin and the possibility that there may be fewer than seven equivalent sites on actin that are saturated by tropomyosin concurrently.

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.

Calcium binding to the regulatory domain of skeletal muscle troponin C induces a highly constrained open conformation.

We have used fluorescence resonance energy transfer to investigate the conformation of the apo and calcium-loaded states of the regulatory N-terminal domain of full-length troponin C mutants from skeletal muscle. The mutants studied each contained a single tryptophan residue (position 22 or 90) and a single cysteine residue (position 52 or 101). The intrinsic fluorophore in each mutant served as an energy donor and the cysteine was conjugated to the acceptor probe 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid. The distributions of two intersite distances (between residues 22 and 52, and residues 90 and 52) were broad in the apo state, indicative of considerable structural dynamics. These distributions were shifted to longer distances and considerably sharpened in the calcium-loaded state. The shifts to longer distances by 8 to 11 A indicate a calcium-induced opening of the N-terminal domain conformation. The transition of the troponin C structure from a closed conformation to an open conformation is accompanied by a substantial reduction of structural fluctuations that dominate in the apo structure as evidenced from the large decrease of the widths of the distributions. This highly constrained open conformation is required as part of the structural basis to facilitate productive interaction between troponin C and troponin I to trigger contraction in skeletal muscle.

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy.

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.

The kinetics of a two-state transition of myosin subfragment 1. A temperature-jump relaxation study.

Temperature-jump measurements were carried out on myosin subfragment 1 (S1) labeled at Cys-707 with 5-(iodoacetamido)fluorescein (S1-AF). The relaxation was monitored by following the increase in the fluorescence intensity of the attached probe after a jump of 5.8 degrees C. A single relaxation process was observed over a range of final temperatures, and the relaxation time decreased from 16.69 ms at 15 degrees C to 3.91 ms at 27 degrees C. The relaxation results are interpreted in terms of a two-state transition: (S1-AF)L K+ in equilibrium with K- (S1-AF)H, and the observed single relaxation time (tau) equals l/(k(+) + k-). The individual first-order rate constants, k+ and k-, were calculated from tau and the equilibrium constant previously determined. The activation energy was 21.9 kcal/mol for the forward reaction and 9.3 kcal/mol for the reverse reaction, corresponding to an enthalpy value of 12.6 kcal/mol for the two-state transition. The results provide, for the first time, direct kinetic evidence of a two-state transition of S1 in the absence of bound nucleotide, and support a two-state model of unliganded myosin subfragment 1.

NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation.

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N-terminal cardiac troponin I proteins, cardiac troponin I(33-80), cardiac troponin I(1-80), cardiac troponin I(1-80)DD and cardiac troponin I(1-80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1-80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N-terminal domain of cardiac troponin I primarily makes contact with the C-terminal domain of cardiac troponin C. The nonphosphorylated cardiac specific amino-terminus, cardiac troponin I(1-80), was found to make additional interactions with the N-terminal domain of cardiac troponin C.