Taipan Natriuretic Peptides Are Potent and Selective Agonists for the Natriuretic Peptide Receptor A.

Cardiovascular ailments are a major cause of mortality where over 1.3 billion people suffer from hypertension leading to heart-disease related deaths. Snake venoms possess a broad repertoire of natriuretic peptides with therapeutic potential for treating hypertension, congestive heart failure, and related cardiovascular disease. We now describe several taipan (Oxyuranus microlepidotus) natriuretic peptides TNPa-e which stimulated cGMP production through the natriuretic peptide receptor A (NPR-A) with higher potencies for the rat NPR-A (rNPR-A) over human NPR-A (hNPR-A). TNPc and TNPd were the most potent, demonstrating 100- and 560-fold selectivity for rNPR-A over hNPR-A. In vivo studies found that TNPc decreased diastolic and systolic blood pressure (BP) and increased heart rate (HR) in conscious normotensive rabbits, to a level that was similar to that of human atrial natriuretic peptide (hANP). TNPc also enhanced the bradycardia due to cardiac afferent stimulation (Bezold-Jarisch reflex). This indicated that TNPc possesses the ability to lower blood pressure and facilitate cardiac vagal afferent reflexes but unlike hANP does not produce tachycardia. The 3-dimensional structure of TNPc was well defined within the pharmacophoric disulfide ring, displaying two turn-like regions (RMSD = 1.15 Å). Further, its much greater biological stability together with its selectivity and potency will enhance its usefulness as a biological tool.

A volumetric and intra-diffusion study of solutions of AlCl3 in two ionic liquids - [C2TMEDA][Tf2N] and [C4mpyr][Tf2N].

Intra-diffusion coefficients (DSi) have been measured for the ionic liquid constituent ions and aluminium-containing species in aluminium chloride (AlCl3) solutions in the ionic liquids 1-(2-dimethyl-aminoethyl)-dimethylethylammonium bis(trifluoromethylsulfonyl)amide ([C2TMEDA][Tf2N]) and N-butyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl)amide ([C4mpyr][Tf2N]), to investigate whether spectroscopically detected interactions between the ions and AlCl3 affect these properties. Such electrolyte solutions are of interest for the electrowinning of aluminium. The temperature, composition and molar volume dependences are investigated. Apparent (Vϕ,1) and partial molar (V1) volumes for AlCl3 have been calculated from solution densities. For [C2TMEDA][Tf2N] solutions, Vϕ,1 increases with increasing solute concentration; for [C4mpyr][Tf2N] solutions, it decreases. In pure [C2TMEDA][Tf2N], the cation diffuses more quickly than the anion, but this changes as the AlCl3 concentration increases. In the [C4mpyr][Tf2N] solutions, the intra-diffusion coefficient ratio remains equal to that for the pure ionic liquid and the aluminium species diffuses at approximately the same rate as the anion at each composition. The intra-diffusion coefficients can be fitted to the Ertl-Dullien free volume power law by superposing the iso-concentration curves with concentration dependent, but temperature independent, molar volume offsets. This suggests that they are primarily dependent on the molar volume and secondarily on a colligative thermodynamic factor due to dilution by AlCl3. AlCl3 complexation by [Tf2N]- and [C2TMEDA]+, confirmed by 27Al, 15N and 19F NMR spectroscopy, seems to play a minor role. Our results indicate that the application of free volume theories might be fruitful in the study of the transport properties of ionic liquid solutions and mixtures.

Molecular details of aluminium-amyloid ß peptide interaction by nuclear magnetic resonance.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that poses major challenges to human health. Both amyloid ß (Aß) and metal ions such as aluminium are implicated in the development of AD. By the means of NMR, the interactions of Al3+ with Aß1-28 peptide as well as the Aß1-28 analogues were studied, and the key binding sites of Al3+ in Aß determined. NMR data showed Al3+ interacts with Aß1-28 at the NH and αH of numerous residues by exhibiting upfield shifts. Using Aß analogues where His6, His13 and His14 were individually replaced by alanine residue(s), including Aß H6A, Aß H13A, Aß H14A, and Aß H6,13,14A, the results demonstrated that the histidine residues (His6, His13 and His14) and N-terminal Asp1 were involved in the Al3+ coordination. These findings provide, for the first time, the details of the molecular interaction between Al3+ and Aß, which points to the potential role of Al3+ in Aß aggregation, hence in AD development.

Applications of WaterControl to TOCSY and COSY experiments.

WaterControl is a solvent suppression method based on WATERGATE and PGSTE and is very efficient in selectively reducing the solvent signal in 1D pulse-acquire and 2D NOESY of protein solutions. In this study, the WaterControl technique was appended to two common 2D NMR methods used in resonance assignment of proteins, namely TOCSY and CLIP-COSY. Similar to that observed in regular 1D pulse-acquire and 2D NOESY, the incorporation of WaterControl in these 2D methods led to excellent solvent suppression superior to that obtained using W3- or W5-based WATERGATE sequences. The water signal was essentially eliminated in the TOCSY and CLIP-COSY with WaterControl while useful cross peaks around the water resonance at ω2 were preserved. This is in contrast to the 2D spectra obtained from the corresponding WATERGATE containing sequences, where these cross peaks in the ω2 region are usually suppressed together with the water resonance. These new WaterControl sequences provide significantly improved water suppression thereby facilitating protein NMR studies.

A Simple and Effective Binomial Block Based Pulse Sequence Capable of Suppressing Multiple NMR Signals.

A binomial-like block based multiple suppression NMR pulse sequence, termed MULTI-GATE-FSB, that is simple to implement with outstanding suppression performance for multiple solvent signals (or multiple resonances) is investigated. The sequence was tested on two water-alcohol solvent systems, and a standard lysozyme sample, with suppression of three or four regions (though it is extendable to any number of regions). The suppression of all solvent signals was possible in the alcohol-water systems tested with both long and short recycle delays and without the requirement for lengthy presaturation pulses. Such a sequence holds promise not only for LC-NMR applications and solvent suppression but for multiple suppression applications in general (e.g., analysis of impurities/components).

NOESY-WaterControl: a new NOESY sequence for the observation of under-water protein resonances.

Highly selective and efficient water signal suppression is indispensable in biomolecular 2D nuclear Overhauser effect spectroscopy (NOESY) experiments. However, the application of conventional water suppression schemes can cause a significant or complete loss of the biomolecular resonances at and around the water chemical shift (ω2). In this study, a new sequence, NOESY-WaterControl, was developed to address this issue. The new sequence was tested on lysozyme and bovine pancreatic trypsin inhibitor (BPTI), demonstrating its efficiency in both water suppression and, more excitingly, preserving water-proximate biomolecular resonances in ω2. The 2D NOESY maps obtained using the new sequence thus provide more information than the maps obtained with conventional water suppression, thereby lessening the number of experiments needed to complete resonance assignments of biomolecules. The 2D NOESY-WaterControl map of BPTI showed strong bound water and exchangeable proton signals in ω1 but these signals were absent in ω2, indicating the possibility of using the new sequence to discriminate bound water and exchangeable proton resonances from non-labile proton resonances with similar chemical shifts to water.

WaterControl: self-diffusion based solvent signal suppression enhanced by selective inversion.

Selective inversion/excitation based solvent signal suppression techniques are widely used in various NMR experiments because of their high efficiency and general applicability. However, these techniques generate a 'null'/suppression region containing (non-quantitatively) degraded solvent and desired resonances because of their reliance on the rejection of the coherence transfer pathway corresponding to all the resonances within the suppression region. To address this issue, the WaterControl technique was developed by inserting a (pulsed gradient - selective inversion pulse - pulsed gradient) unit into each 'transverse' period of a standard stimulated echo pulse sequence so that the coherence transfer pathways corresponding to both the suppression and non-suppression regions can be selected in one transient. The new sequence affords a diffusion based and quantifiable solvent signal suppression with no or minimal loss of features of interest. Copyright © 2016 John Wiley & Sons, Ltd.

Physical characterization using diffusion NMR spectroscopy.

NMR diffusion measurements (or dNMR) provide a powerful tool for analysis of solution organization and microgeometry of the environment by probing random molecular motion. Being a very versatile method, dNMR can be applied to a large variety of samples and systems. Here, a brief introduction into dNMR and a summary of recent advances in the field are presented. The research topics include restricted diffusion, anisotropic diffusion, polymer dynamics, solution structuring and dNMR method development. The dNMR studied systems include plants, cells (cell models), liquid crystals, polymer solutions, ionic liquids, supercooled solutions, untreated water, amino acid solutions and more. It is demonstrated how a variety of dNMR methods can be applied to a system to extract the data on particular structures present among, formed by or surrounding the diffusing particles. It is also demonstrated how dNMR methods can be developed to allow probing larger geometries, low sample concentrations and faster processes. Copyright © 2016 John Wiley & Sons, Ltd.

Insights into hERG K+ channel structure and function from NMR studies.

The unique gating kinetics of hERG K(+) channels are critical for normal cardiac repolarization, and patients with mutations in hERG have a markedly increased risk of cardiac arrhythmias and sudden cardiac arrest. HERG K(+) channels are also remarkably promiscuous with respect to drug binding, which has been a very significant problem for the pharmaceutical industry. Here, we review the progress that has been made in understanding the structure and function of hERG K(+) channels with a particular focus on nuclear magnetic resonance studies of the domains of the hERG K(+) channel.

Restricted diffusion in annular geometrical pores.

Nuclear magnetic resonance (NMR) diffusion (including diffusion MRI) experiments are only as powerful as the models used to analyse the NMR diffusion data. A major problem, especially with measurements on biological systems, is that the existing models are only very poor approximations of cellular shape. Here, diffusion propagators and pulsed gradient spin-echo attenuation equations are derived in the short gradient pulse limit for diffusion within the annular region of a concentric cylinder of finite length and, similarly, within the annular region of a concentric sphere. The models include the possibility of relaxation at the boundaries and, in the case of the concentric cylinder, having the cylinder arbitrarily oriented with respect to the direction of the applied field gradient. The two models are also of interest due to their direct analogy to optical double slit diffraction. Also expressions for the mean square displacements, which are very useful information for determining the diffusion coefficient within these complex geometries, are obtained as well as for the limiting cases of diffusion on cylindrical and spherical shells and in a ring.

Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity.

J-compensated PGSE: an improved NMR diffusion experiment with fewer phase distortions.

Peak distortion caused by homonuclear (1)H J-coupling is a major problem in many spin-echo-based experiments such as pulsed gradient spin-echo (PGSE) experiments. Although peak phase distortions can be lessened by the incorporation of anti-phase purging sequences, the sensitivity is substantially decreased. Techniques for lessening the effect of homonuclear J-coupling evolution in spin-echo-based experiments have been investigated. Two potentially useful candidates include a J-compensated inversion sequence that is efficient over a wide range of J-coupling values and a pulse sequence that refocuses homonuclear J-evolution during the spin-echo. The latter was found to work superbly on samples containing two spin (AX or AB) systems and still provided significant advantage over the standard method on samples containing more complicated spin systems. Implementation of this J-refocusing technique into a PGSE-type experiment (J-PGSE) leads to dramatic improvement of spectra and easier data analysis. The J-PGSE sequence should find applications in many diffusion studies where the PGSE-type method is required and should be a viable alternative to PGSTE especially in dilute samples due to its enhanced sensitivity.

Structure of the pore-helix of the hERG K(+) channel.

The hERG K(+) channel undergoes rapid inactivation that is mediated by 'collapse' of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600-642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two-four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K(+) channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.

Examination of cucurbit[7]uril and its host-guest complexes by diffusion nuclear magnetic resonance.

The self-diffusion of cucurbit[7]uril (CB[7]) and its host-guest complexes in D2O has been examined using pulsed gradient spin-echo nuclear magnetic resonance spectroscopy. CB[7] diffuses freely at a concentration of 2 mM with a diffusion coefficient (D) of 3.07 x 10(-10) m(2) s(-1). At saturation (3.7 mM), CB[7] diffuses more slowly (D = 2.82 x 10(-10) m(2) s(-1)) indicating that it partially self-associates. At concentrations between 2 and 200 mM, CsCl has no effect on the diffusion coefficient of CB[7] (1 mM). Conversely, CB[7] (2 mM) significantly affects the diffusion of 133Cs+ (1 mM), decreasing its diffusion coefficient from 1.86 to 0.83 x 10(-9) m(2) s(-1). Similar changes in the rate of diffusion of other alkali earth metal cations are observed upon the addition of CB[7]. The diffusion coefficient of 23Na+ changes from 1.26 to 0.90 x 10(-9) m(2) s(-1) and 7Li+ changes from 3.40 to 3.07 x 10(-9) m(2) s(-1). In most cases, encapsulation of a variety of inorganic and organic guests within CB[7] decreases their rates of diffusion in D2O. For instance, the diffusion coefficient of the dinuclear platinum complex trans-[[PtCl(NH3)2}2mu-dpzm](2+) (where dpzm is 4,4'-dipyrazolylmethane) decreases from 4.88 to 2.95 x 10(-10) m(2) s(-1) upon encapsulation with an equimolar concentration of CB[7].

PGSTE-WATERGATE: an STE-based PGSE NMR sequence with excellent solvent suppression.

A new stimulated-echo based pulsed gradient spin-echo NMR diffusion sequence incorporating WATERGATE solvent suppression, PGSTE-WATERGATE, is presented. The sequence provides superb solvent suppression without any phase distortions. The sequence is simple to set up and particularly suited to measuring diffusion coefficients in aqueous solution such as is commonly required in pharmaceutical and combinatorial applications. The utility of the sequence is demonstrated on samples containing lysozyme and sucrose. Importantly, the high degree of phase-distortion suppression allows more complicated selective pi pulses to be used to enhance the selectivity of solvent suppression.

NMR diffusion and relaxation studies of 2-nitroimidazole and albumin interactions.

Nitroimidazole derivatives are of current interest in the development of hypoxia targeting agents and show potential in the establishment of quantitative measures of tumor hypoxia. In this study, the binding of 2-nitroimidazole to albumin was probed using NMR diffusion and relaxation measurements. Binding studies were conducted at three different protein concentrations (0.23, 0.30 and 0.38mM) with drug concentrations ranging from 0.005-0.16M at 298K. Quantitative assessments of the binding model were made by evaluating the number of binding sites, n, and association constant, K. These were determined to be 21±3 and 53±4M-1, respectively.

Mammalian l-to-d-amino-acid-residue isomerase from platypus venom.

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.

Apparatus for rapid adjustment of the degree of alignment of NMR samples in aqueous media: verification with residual quadrupolar splittings in (23)Na and (133)Cs spectra.

NMR spectra of (23)Na(+) and (133)Cs(+) in gelatine in a silicone rubber tube that was stretched to various extents showed remarkably reproducible resonance multiplicity. The relative intensities of the components of the split peaks had ratios, 3:4:3, and 7:12:15:16:15:12:7, respectively, that conformed with those predicted using a Mathematica program. The silicone-rubber tube was sealed at its lower end by a small rubber stopper and placed inside a thick-walled glass tube. Gelatine was injected in solution into the silicone tube and 'set' by cooling below 30 degrees C. A plastic thumb-screw held the silicone tube at various degrees of extension, up to approximately 2-fold. After constituting the gel in buffers containing NaCl and CsCl, both (23)Na and (133)Cs NMR spectroscopy revealed that after stretching the initial single Lorentzian line was split into a well-resolved triplet and a heptet, respectively. This was interpreted as being due to coupling between the electric quadrupoles of the nuclei and the average electric field gradient tensor of the collagen molecules of gelatine; these molecules became progressively more aligned in the direction of the main magnetic field, B(0), of the vertical bore magnet, as the gel was stretched. This apparatus provides a simple way of demonstrating fundamental physical characteristics of quadrupolar cations, some characteristics of gelatine under stretching, and a way to invoke static distortion of red blood cells. It should be useful with these and other cell types, for studies of metabolic and membrane transport characteristics that may change when the cells are distorted, and possibly for structural studies of macromolecules.

D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin.

The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an alpha-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin.