Probing the allosteric NBD-TMD crosstalk in the ABC transporter MsbA by solid-state NMR.

The ABC transporter MsbA plays a critical role in Gram-negative bacteria in the regulation of the outer membrane by translocating core-LPS across the inner membrane. Additionally, a broad substrate specificity for lipophilic drugs has been shown. The allosteric interplay between substrate binding in the transmembrane domains and ATP binding and turnover in the nucleotide-binding domains must be mediated via the NBD/TMD interface. Previous studies suggested the involvement of two intracellular loops called coupling helix 1 and 2 (CH1, CH2). Here, we demonstrate by solid-state NMR spectroscopy that substantial chemical shift changes within both CH1 and CH2 occur upon substrate binding, in the ATP hydrolysis transition state, and upon inhibitor binding. CH2 is domain-swapped within the MsbA structure, and it is noteworthy that substrate binding induces a larger response in CH2 compared to CH1. Our data demonstrate that CH1 and CH2 undergo structural changes as part of the TMD-NBD cross-talk.

Comparative evaluation of approach to cardiovascular care in neonatal encephalopathy undergoing therapeutic hypothermia.

OBJECTIVE: To analyze the association between cardiovascular care and adverse outcome in infants undergoing therapeutic hypothermia for neonatal encephalopathy (NE). STUDY DESIGN: This was a retrospective cohort study of 176 infants with NE and hypotension, admitted to the SickKids Hospital (Center A, n = 86) or Semmelweis University (Center B, n = 90). RESULT: The lowest systolic/diastolic blood pressures were comparable amongst centers; however, proportion of cardiovascular support was lower in Center A (51% vs 97% in Center B). Overall rate of death or abnormal MRI (adverse outcome) were comparable between centers, although pattern differed with more basal ganglia injury in Center B. A 24-hour longer duration of cardiovascular support increased the odds for adverse outcome by 14%. CONCLUSION: We demonstrated that management of hemodynamic instability in infants with NE was markedly different in two high-volume NICUs and showed that longer duration of cardiovascular medication is an independent risk factor for adverse outcome.

Neonatal encephalopathy therapy optimization for better neuroprotection with inhalation of CO2: the HENRIC feasibility and safety trial.

BACKGROUND: There is an association between hypocapnia and adverse neurodevelopmental outcome in infants with neonatal encephalopathy (NE). Our aim was to test the safety and feasibility of 5% CO2 and 95% air inhalation to correct hypocapnia in mechanically ventilated infants with NE undergoing therapeutic hypothermia. METHODS: Ten infants were assigned to this open-label, single-center trial. The gas mixture of 5% CO2 and 95% air was administered through patient circuits if the temperature-corrected PCO2 ≤40 mm Hg. The CO2 inhalation was continued for 12 h or was stopped earlier if the base deficit (BD) level decreased <5 mmol/L. Follow-up was performed using Bayley Scales of Infant Development II. RESULTS: The patients spent a median 95.1% (range 44.6-98.5%) of time in the desired PCO2 range (40-60 mm Hg) during the inhalation. All PCO2 values were >40 mm Hg, the lower value of the target range. Regression modeling revealed that BD and lactate had a tendency to decrease during the intervention (by 0.61 and 0.55 mmol/L/h, respectively), whereas pH remained stable. The rate of moderate disabilities and normal outcome was 50%. CONCLUSIONS: Our results suggest that inhaled 5% CO2 administration is a feasible and safe intervention for correcting hypocapnia.

Neurodevelopmental effect of intracranial hemorrhage observed in hypoxic ischemic brain injury in hypothermia-treated asphyxiated neonates - an MRI study.

BACKGROUND: Identification of early signs of hypoxic ischemic encephalopathy (HIE) with magnetic resonance imaging (MRI) has proven of prognostic significance. Yet, the importance of intracranial hemorrhage (ICH), being present concomitantly had not been investigated yet, despite the known influence of hypothermia on hemostasis. We aimed to determine whether presence of ICH on MRI alongside the signs of HIE have an impact on prognosis in neonates with the clinical diagnosis of HIE. METHODS: A retrospective study of consecutively sampled 108 asphyxiated term infants admitted to a tertiary neonatal intensive care unit (between 2007 and 2016), treated with whole body hypothermia and having brain MRI within 1 week of life was conducted. Presence or absence of HIE signs on MRI (basal ganglia-thalamus, watershed pattern and total brain injury) and on MR spectroscopy (lactate peak with decreased normal metabolites measured by Lac/NAA ratio) and/or of the five major types of ICH were recorded. Neurodevelopmental outcome was measured with Bayley Scales of Infant Development-II (BSID-II) test. Death or abnormal neurodevelopment (BSID-II score < 85) was defined as poor outcome in Chi-square test. Multivariate logistic regression analysis was performed on survivors. RESULTS: MRI and MR-spectroscopy (MRS) signs of HIE were present in 72% (n = 78). 36% (n = 39) of neonates had ICH, being mainly small in size. Chi-square test showed a relationship between neurodevelopmental outcome and initial MRI. Unadjusted logistic regression showed that neonates presenting MRI and MRS signs of HIE have 6.23 times higher odds for delayed mental development (OR = 6.2292; CI95% = [1.2642; 30.6934], p = 0.0246), than infants without imaging alterations; with no ICH effect on outcome. Adjustment for clinical and imaging parameters did not change the pattern of results, i.e. HIE remained an independent risk factor for delayed neurodevelopment (OR = 6.2496; CI95% = [1.2018; 32.4983], p = 0.0294), while ICH remained to have no significant effect. CONCLUSION: HIE related MRI abnormalities proved to be important prognostic factors of poor outcome in cooled asphyxiated infants when present, suggesting that early MRI with MRS is beneficial for prognostication. Interestingly, ICHs present in about one third of all cases had no significant effect on neurodevelopmental outcome, despite the known hemostasis altering effects of hypothermia.

Prognostic value of early, conventional proton magnetic resonance spectroscopy in cooled asphyxiated infants.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) commonly leads to neurodevelopmental impairment, raising the need for prognostic tools which may guide future therapies in time. Prognostic value of proton MR spectroscopy (H-MRS) between 1 and 46 days of age has been extensively studied; however, the reproducibility and generalizability of these methods are controversial in a general clinical setting. Therefore, we investigated the prognostic performance of conventional H-MRS during first 96 postnatal hours in hypothermia-treated asphyxiated neonates. METHODS: Fifty-one consecutive hypothermia-treated HIE neonates were examined by H-MRS at three echo-times (TE = 35, 144, 288 ms) between 6 and 96 h of age, depending on clinical stability. Patients were divided into favorable (n = 35) and unfavorable (n = 16) outcome groups based on psychomotor and mental developmental index (PDI and MDI, Bayley Scales of Infant Development II) scores (≥ 70 versus < 70 or death, respectively), assessed at 18-26 months of age. Associations between 36 routinely measured metabolite ratios and outcome were studied. Age-dependency of metabolite ratios in whole patient population was assessed. Prognostic performance of metabolite ratios was evaluated by Receiver Operating Characteristics (ROC) analysis. RESULTS: Three metabolite ratios showed significant difference between outcome groups after correction for multiple testing (p < 0.0014): myo-inositol (mIns)/N-acetyl-aspartate (NAA) height, mIns/creatine (Cr) height, both at TE = 35 ms, and NAA/Cr height at TE = 144 ms. Assessment of age-dependency showed that all 3 metabolite ratios (mIns/NAA, NAA/Cr and mIns/Cr) stayed constant during first 96 postnatal hours, rendering them optimal for prediction. ROC analysis revealed that mIns/NAA gives better prediction for outcome than NAA/Cr and mIns/Cr with cut-off values 0.6798 0.6274 and 0.7798, respectively, (AUC 0.9084, 0.8396 and 0.8462, respectively, p < 0.00001); mIns/NAA had the highest specificity (95.24%) and sensitivity (84.62%) for predicting outcome of neonates with HIE any time during the first 96 postnatal hours. CONCLUSIONS: Our findings suggest that during first 96 h of age even conventional H-MRS could be a useful prognostic tool in predicting the outcome of asphyxiated neonates; mIns/NAA was found to be the best and age-independent predictor.

Novel structured MRI reporting system in neonatal hypoxic-ischemic encephalopathy - issues of development and first use experiences.

BACKGROUND AND PURPOSE: To develop an evidence-based, standardized structured reporting (SR) method for brain MRI examinations in neonatal hypoxic-ischemic encephalopathy (HIE) suitable both for clinical and research use. METHODS: SR template development was based on comprehensive review of the pertinent literature with the basic sections and subdivisions of the template defined according to MRI sequences (both conventional and diffusion-weighted, MR-spectroscopy (MRS), and T2*-weighted imaging), and the items targeted on age-related imaging patterns of HIE. In order to evaluate the usability of the proposed SR template we compared data obtained from the brain MR image analysis of 87 term and 19 preterm neonates with the literature. The enrolled 106 infants were born between 2013 and 2015, went through therapeutic hypothermia according to the TOBY criteria due to moderate to severe asphyxia and had at least one brain MRI examination within the first two weeks of life. Ethical approval was obtained for this retrospective study. Descriptive statistical analysis was also performed on data exported from the structured reporting system as feasibility test. RESULTS: The mean gestational age of the study population was 38.3±2.2 weeks; brain MRI was performed on 5.8±2.9 day of life, hence in 78% of our patients after the conclusion of therapeutic hypothermia. Our main imaging findings were concordant to the pertinent literature. Moreover, we identified a characteristic temporal evolution of diffusion changes. Interestingly 18% (n=19/106) of the clinically asphyxiated infants had isolated axial-extraaxial haemorrhage without any imaging sign of HIE. CONCLUSION: In this article our approach of reporting HIE cases with our novel SR template is described. The SR template was found suitable for reporting HIE cases, moreover it uncovered time and location dependent evolution of diffusion abnormalities (and pseudonormalization, as well), suggesting its usefulness in clinical research applications. The high number of isolated intracranial haemorrhages, and the changing diffusion pattern emphasizes the importance of early imaging in HIE.

Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin.

The UV light absorbing species of proteorhodopsin with deprotonated Schiff base retinal was investigated using steady-state fluorescence and femtosecond pump-probe spectroscopy. Compared to the all-trans retinal with protonated Schiff base, the deprotonated chromophore absorbs at 365 nm and exhibits a blue-shifted fluorescence spectrum. The unusually long-lived excited state decays bi-exponentially with time constants of 8 ps and 130 ps to form a deprotonated 13-cis retinal as the primary photo-product.

The ABC exporter MsbA probed by solid state NMR ­ challenges and opportunities.

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.

Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy.

Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and a slight alteration of the retinal chain's out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S0-S1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump-probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.

Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR.

Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP(+) and MTP(+) to EmrE reconstituted into 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes by (31)P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between (13)C-labeled TPP(+) and key residues within the EmrE dimer has been probed by through-space (13)C-(13)C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such as TPP(+) and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection.

How to investigate interactions between membrane proteins and ligands by solid-state NMR.

Solid-state NMR is an established method for biophysical studies of membrane proteins within the lipid bilayers and an emerging technique for structural biology in general. In particular magic angle sample spinning has been found to be very useful for the investigation of large membrane proteins and their interaction with small molecules within the lipid bilayer. Using a number of examples, we illustrate and discuss in this chapter, which information can be gained and which experimental parameters need to be considered when planning such experiments. We focus especially on the interaction of diffusive ligands with membrane proteins.

Nimustine hydrochloride: the first crystal structure determination of a 2-chloroethyl-N-nitrosourea hydrochloride derivative by X-ray powder diffraction and solid-state NMR.

Nimustine hydrochloride [systematic name: 4-amino-5-({[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino}methyl)-2-methylpyrimidin-1-ium chloride], C(9)H(14)ClN(6)O(2)(+)·Cl(-), is a prodrug of CENU (chloroethylnitrosourea) and is used as a cytostatic agent in cancer therapy. Its crystal structure was determined from laboratory X-ray powder diffraction data. The protonation at an N atom of the pyrimidine ring was established by solid-state NMR spectroscopy.

Histidine-rich branched peptides as Cu(II) and Zn(II) chelators with potential therapeutic application in Alzheimer's disease.

Two histidine-rich branched peptides with one lysine as a branching unit have been designed and synthesized by solid-phase peptide synthesis. Their complex formation with Cu(II) and Zn(II) as well as their ability to attenuate the metal-ion induced amyloid aggregation has been characterized. Both peptides can keep Cu(II) and Zn(II) in complexed forms at pH 7.4 and can bind two equivalents of metal ions in solutions with excess metal. The stoichiometry, stability and structure of the complexes formed have been determined by pH potentiometry, UV-Vis spectrophotometry, circular dichroism, EPR and NMR spectroscopy and ESI-MS. Both mono- and bimetallic species have been detected over the whole pH range studied. The basic binding mode is either a tridentate {N(amino), N(amide), N(im)} or a histamine-type of coordination which is complemented by the binding of far imidazole or amino groups leading to macrochelate formation. The peptides were able to prevent Cu(II)-induced Aß(1-40) aggregation but could not effectively compete for Zn(II) in vitro. Our results suggest that branched peptides containing potential metal-binding sites may be suitable metal chelators for reducing the risk of amyloid plaque formation in Alzheimer's disease.

Two pyridine derivatives as potential Cu(II) and Zn(II) chelators in therapy for Alzheimer's disease.

Two pyridine derivatives, DMAP and ENDIP, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. Their complex formation with Cu(ii) and Zn(ii) were characterised in detail. In the case of ENDIP a high stability tetradentate ML complex is formed at physiological pH both with Cu(ii) and Zn(ii). DMAP was found to be a weaker metal binder. At physiological pH, it forms a bidentate ML complex with Zn(ii) and MLH(-1) and ML(2) complexes with Cu(ii), depending on the metal ion to ligand ratio. Fluorescence spectroscopy and dynamic light scattering measurements proved that ENDIP effectively competes with aggregated amyloid-beta peptides (Abeta) for both Cu(ii) and Zn(ii) and thus is able to prevent the metal ion-induced amyloid aggregation and to resolubilise amyloid precipitates.

A pH-metric, UV, NMR, and X-ray crystallographic study on arsenous acid reacting with dithioerythritol.

The aqueous solutions of arsenous acid with the meso and racemic forms of 1,4-dithiol-butane-2,3-diol, namely, dithioerythritol (dte) and dithiothreitol (dtt), respectively, were titrated pH-metrically in different molar ratios. The p K a values determined for As(OH) 3, and dtt were in good accordance with the literature data, and we determined for the first time the p K a value of dte. The deprotonation steps of both M (As(OH) 3 considered as a central metal ion) and H 2L components dte and dtt (considered as ligands) appeared at a higher pH in the titration curves of the ternary systems (M, H 2L, H (+)) than in the individual component. This unusual observation is explained by the condensation reactions between the reagents taking place in the pH < 8 range. In the solutions of c As(III) > 5.10 (-3) M, the precipitate formed upon mixing the arsenous acid and H 2L solutions in neutral medium, and the formation of the precipitate shifted toward acidic pH on the increase of the total concentrations. This indicated that pH-metry can follow the reactions only in an indirect way. Useful, but not satisfactory, information can be obtained by means of this method alone. Combined with NMR and UV spectroscopic measurements it is revealed that depending on the As(III)/H 2L molar ratio, different complexes form in the solutions. In the species with 1:2 composition, one of the ligands is strongly bound to the arsenic(III) probably via its two thiolate, while the second one is attached only weakly. The crystal structure of an As(III)-dte crystal of 1:1 composition, grown from ethanolic solution, shows that As(III) binds the ligand through its three p-orbitals in a manner similar to that expected in aqueous solution. While the uptake of the second ligand cannot be detected by pH-metry, the decomposition of thioether bonds above pH approximately 10 is confirmed by the change in UV spectra at approximately 265 nm to be a base-consuming process. In such alkaline solutions, most probably, rearrangement of the bonding scheme occurs, resulting in ligands being bound to the arsenic(III) through the oxygen donor atoms.

Interactions of insulin-mimetic zinc(II) complexes with cell constituents: glutathione and ATP.

Ternary complex formation of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 3-hydroxy-1,2-dimethyl-pyridinone with (O,O), 2-picolinic acid and 6-methylpicolinic acid with (N,O) and the tridentate 2,6-dipicolinic acid with (O,N,O) coordination modes was studied in aqueous solutions by pH-potentiometry and spectroscopic (UV, CD, ESI-MS) methods in the presence of critical cell constituents such as L-glutathione reduced (GSH) and adenosine 5'-triphosphate (ATP). Results showed that formation of the ternary complexes was hindered in the case of 2,6-dipicolinic acid, especially with ATP, while it was favoured with the bidentate ligands in the physiological pH range. Driving force of the formation of mixed-ligand species was found to be a more enhanced coordination of GSH and ATP as second ligands in the ternary complexes than in their binary ones due to steric and electrostatic reasons. The mitochondrial dehydrogenase activity of the zinc(II) complexes, as an indirect indicator for the glucose intake, was measured on Mono Mac and 3T3-L1 adipocyte cell lines. The activity of the complexes up to approximately 10-100 microM concentration was in the range of the effect of 0.75-1.5 microM insulin, while at higher concentration it was broken down due to the sensitivity of the cells to toxicity of the complexes.

Endomorphin-2, an endogenous tetrapeptide, protects against Abeta1-42 in vitro and in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the beta-amyloid aggregates formed mainly by Abeta1-42 peptide. Protective pentapeptides [e.g., Leu-Pro-Phe-Phe-Asp (LPFFD)] have been shown to prevent neuronal toxicity of Abeta1-42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin-2 (End-2, amino acid sequence: YPFF), defends against Abeta1-42 induced neuromodulatory effects at the cellular level. Although End-2 does not interfere with the kinetics of Abeta fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Abeta1-42 during aggregation, as revealed by tritium-labeled End-2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Abeta1-42 in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide (MTT) assay. In vitro and in vivo electrophysiological experiments show that End-2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA-evoked response-enhancing effect of Abeta1-42. Studies using [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, show that the protective effects of the tetrapeptide are not mu-receptor modulated. The endogenous tetrapeptide End-2 may serve as a lead compound for the drug development in the treatment of AD.

Al(III)-binding ability of an octapeptide and its phosphorylated derivative.

A synthetic octapeptide, H-GlyGluGlyGluGlySerGlyGly-OH, and its phosphorylated Ser derivative were synthetized and their solution speciation and binding modes in their complexes with Al(III) were measured. One goal of the work was find a lead compound for the design of a selective peptide-based Al(III) chelator. pH-potentiometry was used to characterize the stoichiometry and the stability of the species formed in the interactions of the metal ion and the peptides, while multinuclear NMR was applied to characterize the binding sites of the metal ion in the complexes. CD spectroscopy revealed a difference in the conformational behaviour of the phosphorylated peptide as compared with its non-phosphorylated parent derivative. The Al(III) is presumed to enhance aggregation through the -PO3H(-)-Al(3+)-PO3(2-)-Al(3+)- intermolecular bindings between the peptide chains. The results of molecular dynamics calculations supported the experimentally obtained secondary structures and the binding position of Al(III).

AlIII ion complexes of saccharic acid and mucic acid: a solution and solid-state study.

The Al(III)-binding abilities of two aldaric acids, D-saccharic acid and mucic acid (the neutral form is denoted as H(2)L), were studied in solution by means of pH potentiometric, (1)H and (13)C NMR, and ESI-MS techniques. The most probable conformations and isomeric binding modes of the complexes formed in solution were determined by density functional theory (DFT) calculations. A solid D-saccharic acid complex K(2)[[Al(LH(-2))(H(2)O)](2)].H(2)O was isolated and crystallographically characterised. The two alcoholic hydroxy groups alpha to the terminal COO(-) groups were found to take part in the coordination, but in different ways. One of them coordinates in a bridging mode. Detailed ESI-MS and NMR studies proved that the complex retains its structure in solution. However, depending on the ligand and the pH, such complexes may exist in two isomeric forms. DFT calculations on the ion [[Al(LH(-2))(H(2)O)](2)](2-) revealed that several orbitals participate in stabilizing the dimeric arrangement.