Cooperative polymerization of photosynthetic pigments in formamide-water solution

The aggregation of bacteriochlorophyll a and bacteriopheophytin a into large oligomers with maximum optical absorption at 860 nm was studied in a 3:1 (vol/vol) formamide/water solution, using optical absorption spectroscopy and electron microscopy. The aggregation is cooperative and proceeds according to two equilibrium constants. Initially, two pigment molecules form a “seed” that absorbs at ≈860 nm. The equilibrium constant, K_a, governing this reaction equals 1.3 × 10³ M^-1 in the case of bacteriochlorophyll a (due to experimental limitations, K_a for bacteriopheophytin a could not be determined). The addition of monomers to aggregates consisting of two or more units is governed by an equilibrium constant, K_b, equal to 2.2 × 10⁶ M^-1 for bacteriochlorophyll a and ≈ 10⁹ M^-1 for bacteriopheophytin a. The enthalpy and entropy changes that drive the bacteriochlorophyll oligomer formation are -9.25 and ≈0.0 kcal/mol, respectively. Above a threshold concentration, the amount of oligomers remains constant but their length continues to increase. Each oligomer appears to consist of dimers that are associated by hydrophobic interactions among their alcohol residues, forming long strands. Single strands presumably coil into helices that are seen as cylinders. The bacteriochlorophyll a oligomers form cylinders with a constant diameter of 150 Å and an average length of 2,000 Å (at 1.5 × 10^-5 M bacteriochlorophyll a). These cylinders contain 200-250 bacteriochlorophyll a dimers. The bacteriopheophytin oligomers coil into wider cylinders (≈400 Å in diameter) which contain ≈600-700 bacteriopheophytin a dimers. In both cases, the separation between the dimers is ≈20 Å. At such distances, the dipolar interactions among adjacent dimers are negligible and do not affect the optical absorption of each individual pair. Therefore, the optical absorption of these pairs can be a tool for investigating the absorption pattern of photosynthetic pigments in vivo.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a

Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK_a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK_a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k₁, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK_a. The inference that k₁ is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK_a is that of the general acid and the higher pK_a is that of the general base.     

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pK_a of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pK_a 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pK_a, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pK_a from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pK_a from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325 cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pK_a shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3 kcal/mol to set the enzyme into a catalytically competent form.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

Arginine: Its pKa value revisited

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pK_a value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pK_a value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pK_a value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.     

Origin of the pKa shift of the catalytic lysine in acetoacetate decarboxylase

The pK_a value of Lys115, the catalytic residue in acetoacetate decarboxylate, was calculated using atomic coordinates of the X-ray crystal structure with consideration of the protonation states of all titratable sites in the protein. The calculated pK_a value of Lys115 (pK_a(Lys115)) was unusually low (≈6) in agreement with the experimentally measured value. Although charged residues impact pK_a(Lys115) considerably in the native protein, the significant pK_a(Lys115) downshift in the protein with respect to aqueous solution was mainly due to loss of the solvation energy in the catalytic active site relative to bulk water.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

Aggregation of ferrihaems. Dimerization and protolytic equilibria of protoferrihaem and deuteroferrihaem in aqueous solution

1. The absorption spectra of deutero- and proto-ferrihaem in aqueous solution at 25°C show marked changes with concentration and pH in the Soret band region. Quantitative studies of these phenomena imply that they are associated with ferrihaem dimerization and with protolytic equilibria involving monomeric (M) and dimeric (D) ferrihaem species according to the scheme: [Formula: see text] 2. For deuteroferrihaem we obtain K=1.9×10⁻², pK_a(M)=7.1, pK_a(D)=7.4. Protoferrihaem has a much higher dimerization constant, K=4.5 and pK_a(D)=7.5 (pK_a(M) is not accessible). 3. Possible structural relationships between monomeric and dimeric ferrihaem species in solution are discussed in relation to recent work on the oxo-bridged nature of crystalline ferrihaem dimers.     

Modulation of an Active-Site Cysteine pKa Allows PDI to Act as a Catalyst of both Disulfide Bond Formation and Isomerization

Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond reduction and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pK_a values of the active-site cysteines of PDI. Here, we examined the role of arginine 120 in modulating the pK_a values of these cysteines. We find that arginine 120 plays a significant role in modulating the pK_a of the C-terminal active-site cysteine in the a domain of PDI and plays a role in determining the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pK_a. Mutation of arginine 120 and the corresponding residue, arginine 461, in the a′ domain severely reduces the ability of PDI to catalyze disulfide bond formation and reduction but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pK_a of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidation and isomerization reactions.     

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK_a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK_a values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK_a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK_a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N^ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK_a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK_a values are governed by short-range polar and Coulomb interactions.     

Association of plasma sRAGE,but not esRAGE with lung function impairment in COPD

Rationale

Plasma soluble Receptor for Advanced Glycation End Product (sRAGE) is considered as a biomarker in COPD. The contribution of endogenous sRAGE (esRAGE) to the pool of plasma sRAGE and the implication of both markers in COPD pathogenesis is however not clear yet. The aim of the current study was therefore to measure plasma levels of esRAGE comparative to total sRAGE in patients with COPD and a control group. Further, we established the relations of esRAGE and total sRAGE with disease specific characteristics such as lung function and D_LCO, and with different circulating AGEs.

Methods

Plasma levels of esRAGE and sRAGE were measured in an 88 patients with COPD and in 55 healthy controls. FEV₁ (%predicted) and FEV₁/VC (%) were measured in both groups; D_LCO (%predicted) was measured in patients only. In this study population we previously reported that the AGE N^ϵ-(carboxymethyl) lysine (CML) was decreased, N^ϵ-(carboxyethyl) lysine (CEL) increased and pentosidine was not different in plasma of COPD patients compared to controls.

Results

Plasma esRAGE (COPD: 533.9 ± 412.4, Controls: 848.7 ± 690.3 pg/ml; p = 0.000) was decreased in COPD compared to controls. No significant correlations were observed between plasma esRAGE levels and lung function parameters or plasma AGEs. A positive correlation was present between esRAGE and total sRAGE levels in the circulation. Confirming previous findings, total sRAGE (COPD: 512.6 ± 403.8, Controls: 1834 ± 804.2 pg/ml; p < 0.001) was lower in patients compared to controls and was positively correlated FEV₁ (r = 0.235, p = 0.032), FEV₁/VC (r = 0.218, p = 0.047), and D_LCO (r = 0.308, p = 0.006). sRAGE furthermore did show a significant positive association with CML (r = 0.321, p = 0.003).

Conclusion

Although plasma esRAGE is decreased in COPD patients compared to controls, only total sRAGE showed a significant and independent association with FEV₁, FEV₁/VC and D_LCO, indicating that total sRAGE but not esRAGE may serve as marker of COPD disease state and severity.     

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (K_{m(app)_NH3} ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (K_{m(app)_NO2}- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.Subject terms: Bacterial genomics, Environmental microbiology, Bacterial physiology     

The pKa Values of Acidic and Basic Residues Buried at the Same Internal Location in a Protein Are Governed by Different Factors

The pK_a values of internal ionizable groups are usually very different from the normal pK_a values of ionizable groups in water. To examine the molecular determinants of pK_a values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK_a, whereas Asp38 and Glu38 titrate with elevated pK_a values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK_a of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK_a values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK_a values of internal groups in proteins. This study suggests that improvements to computational methods for pK_a calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.     

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H⁺ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK_a values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK_a values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK_a values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

Structure–activity relationship,in vitro and in vivo evaluation of novel dienyl sulphonyl fluorides as selective BuChE inhibitors for the treatment of Alzheimer's disease

To discover novel scaffolds as leads against dementia, a series of δ-aryl-1,3-dienesulfonyl fluorides with α-halo, α-aryl and α-alkynyl were assayed for ChE inhibitory activity, in which compound A10 was identified as a selective BuChE inhibitor (IC₅₀ = 0.021 μM for eqBChE, 3.62 μM for hBuChE). SAR of BuChE inhibition showed: (i) o- > m- > p-; –OCH₃ > –CH₃ > –Cl (–Br) for δ-aryl; (ii) α-Br > α-Cl, α-I. Compound A10 exhibited neuroprotective, BBB penetration, mixed competitive inhibitory effect on BuChE (K_i = 29 nM), and benign neural and hepatic safety. Treatment with A10 could almost entirely recover the Aβ_1-42-induced cognitive dysfunction to the normal level, and the assessment of total amount of Aβ_1-42 confirmed its anti-amyloidogenic profile. Therefore, the potential BuChE inhibitor A10 is a promising effective lead for the treatment of AD.     

CMR-derived TAPSE measurement: a semi-quantitative method of right ventricular function assessment in patients with hypertrophic cardiomyopathy

Aim

To compare cardiovascular magnetic resonance (CMR)-derived right ventricular fractional shortening (RVFS), tricuspid annular plane systolic excursion with a reference point within the right ventricular apex (TAPSE_in) and with one outside the ventricle (TAPSE_out) with the standard volumetric approach in patients with hypertrophic cardiomyopathy (HCM).

Methods and results

105 patients with HCM and 20 healthy subjects underwent CMR. In patients with HCM, TAPSE_in (r = 0.31, p = 0.001) and RVFS (r = 0.35, p = 0.0002) revealed a significant but weak correlation with right ventricular ejection fraction (RVEF), whereas TAPSE_out (r = 0.57, p < 0.0001) showed a moderate correlation with RVEF. The ability to predict RVEF < 45 % in HCM patients was best for TAPSE_out. In patients with hypertrophic obstructive cardiomyopathy (HOCM), RVEF showed a significant but weak correlation with TAPSE_out (r = 0.36, p = 0.02) and no correlation with TAPSE_in (r = 0.05, p = 0.07) and RVFS (r = 0.02, p = 0.2). In patients with hypertrophic non-obstructive cardiomyopathy (HNCM), there was a moderate correlation between RVEF and TAPSE_out (r = 0.57, p < 0.0001) and a weak correlation with TAPSE_in (r = 0.39, p = 0.001) and RVFS (r = 0.38, p = 0.002). In the 20 healthy controls, there was a strong correlation between RVEF and all semi-quantitative measurements.

Conclusion

CMR-derived TAPSE_in is not suitable to determine right ventricular function in HCM patients. TAPSE_out showed a good correlation with RVEF in HNCM patients but only a weak correlation in HOCM patients. TAPSE_out might be used for screening but the detection of subtle changes in RV function requires the 3D volumetric approach.     

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO₂ to bicarbonate and protons, with a k_cat value of 3.0 × 10⁵ s⁻¹ and k_cat/K_M value of 3.9 × 10⁷ M⁻¹ s⁻¹. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (K_I values of 6.2–94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (K_I values of 0.71–0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed K_I values in the range of 3.1–9.3 mM.     

Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis

Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (K_i: 0.1, 0.4, 6.6 nM) resembled pepstatin A (K_i: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (K_i: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (K_i: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.