A method for identification of inhibitors of the phosphorylation reactions of bacterial response regulator proteins using (31)P nuclear magnetic resonance spectroscopy.

Bacterial response regulators are attractive targets for antibacterial drug development, yet random screening against these targets has failed as yet to identify chemicals that constitute viable leads. Alternative methods to provide leads for drug development based on identification and optimization of low affinity ligands from NMR screens have been described. However, leads from these processes still require verification in a bioassay, which is often problematic if compounds have unfavorable optical and solubility properties. A simple method, based on using NMR to observe the activity of the target, is described. It has the advantages of being able to characterize both low affinity leads and a wider selection of compounds in a structure activity relationships series, without the problems affecting a fluorescence assay. In this example we use (31)P to monitor the turnover of a bacterial response regulator, but the generic approach could be applied to other nuclei and thus a range of biological systems.

Conformation of the cytoplasmic domain of phospholamban by NMR and CD.

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

A spectroscopic study of the mitochondrial transit peptide of rat malate dehydrogenase.

A peptide corresponding to the N-terminal sequence of the rat malate dehydrogenase, comprising the transit sequence and two residues of the mature protein (MLSALARPVGAALR-RSFSTSAQNNAK) has been chemically synthesized, and its structural characteristics investigated by Fourier-transform i.r. (FT-IR), c.d. and 1H-n.m.r. spectroscopy. FT-IR and c.d. spectra of the peptide were recorded in a variety of environments (aqueous solution, trifluoroethanol) and after incorporation into phospholipid bilayers. The peptide was found to be mainly in aperiodic or undefined conformation in aqueous solution. However, in trifluoroethanol a marked increase in alpha-helical content was observed. An increase in alpha-helical content was also observed in negatively charged lipids (dimyristoylphosphatidylglycerol and cardiolipin). However, when reconstituted in a zwitterionic phospholipid (dimyristoylphosphatidylcholine), no alpha-helical structure was observed. N.m.r. spectroscopy was used to characterize the helical structure in greater detail in trifluoroethanol. The 1H-n.m.r. spectrum of the peptide in this solvent was assigned using standard homonuclear two-dimensional methods. The observed patterns of nuclear Overhauser enhancements confirmed the deductions obtained from c.d. and FT-1R spectroscopy concerning the solution conformation, suggesting a region of flexible nascent helix between Ala-4 and Ser-18. This structure is discussed in terms of the possible function of the peptide.

An electrophysiological and spectroscopic study of the properties and structure of biological calcium channels. Investigations of a model ion channel.

The N- and C-terminally protected peptide N-acetyl-Asp-Phe-Ala-Asn-Arg-Val-Leu-Leu-Ser-Leu-Phe-Thr-Ile-Glu-Met-Leu -Leu-Lys-Met-Leu-NH2, closely based on the sequence of the putative S2 membrane spanning helix of domain II of the dihydropyridine receptor calcium channel of the T-system of skeletal muscle, residues 465-486 (Tanabe et al. (1987) Nature 328, 313-318) has been synthesised. Conductance measurements in planar lipid bilayers show that the peptide is capable of inducing the transmembrane passage of calcium and barium ions, in preference to monovalent cations. No anion conductance is observed. 1H-NMR spectroscopy demonstrates that in an amphilic solvent, methanol, the peptide forms highly stable structures characterised by very slow exchange with solvent of peptide N-H protons. Double-quantum filtered phase-sensitive COSY shows that, on the basis of NH-CH alpha scalar coupling constants, most peptide torsion angles are appropriate to an overall alpha-helical conformation; the presence of some alpha-helix is also supported by CD measurements. Most side-chain connectivities have been identified in a DIPSI-TOCSY experiment. This evidence has been used to construct a low-resolution model of the ion-conducting channel of the muscle T-system dihydropyridine receptor from the sequences of the four homologous putative channel-lining stretches. It is characterised by an association of acidic residues at the putative extra-membranous face of the channel, followed by a predominantly hydrophobic band. The next prominent feature of the model is an ordered array of four acidic residues (glutamates 100, 478, 846 and 1164), followed by four lysines (104, 482, 850 and 1168) which may play a gating role.

Spectroscopic and physicochemical studies on the interactions of reversible H+/K(+)-ATPase inhibitors with phospholipid bilayers.

Three complementary techniques, differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, have been used to characterise the interactions between dimyristoylphosphatidylcholine (DMPC) model biological membranes and two non-covalent inhibitors of the gastric (H+, K+)-ATPase. DSC, FT-IR and deuterium NMR studies of side-chain perdeuterated DMPC (DMPC-d54) support the prediction, based on physical property measurements, that SK&F 96079 partitions readily into phospholipid bilayers, resulting in a slight but measurable disordering of the lipid hydrocarbon side-chain motion and a concomitant reduction in the co-operativity and onset temperature of the gel to liquid crystalline phase transition. However, FT-IR and deuterium NMR studies show that the bilayer structure remains intact even at high (1:4) compound to lipid molar ratios. Proton (1H) NMR nuclear Overhauser effect determinations in sonicated codispersions reveal details of the membrane bound conformations of SK&F 96079. The structurally related analogue SK&F 96464, also studied by 1H-NMR, can be shown, by interpreting the effects of nitroxide-labelled fatty acid relaxation probes, to adopt a well-defined orientation relative to the bilayer, in contrast to SK&F 96079. This orientation directs the proton at the 5-position of the quinoline ring towards the hydrophobic centre of the bilayer, and the quinoline 8-methoxy group towards the surface and hence the aqueous phase. Molecular modelling has been used to rationalise this orientation in terms of hydrogen bonds between the amino NH group of SK&F 96464 and the sn-1 carbonyl group of DMPC, and between the NH group of the protonated quinoline ring of SK&F 96464 and the DMPC phosphodiester group.

A proton nuclear magnetic resonance and molecular modeling study of calmidazolium (R24571) binding to calmodulin and skeletal muscle troponin C.

1H NMR spectroscopy at 360 MHz has been used to study the interactions between the calmodulin function inhibitor calmidazolium (R24571) and (i) calmodulin (CaM) and (ii) skeletal muscle troponin C (sTnC). One equivalent of racemic calmidazolium binds tightly to CaM and perturbs a number of protein signals, corresponding to residues in both dicalcium-binding domains, in a manner characteristic of slow exchange. Calmidazolium binds with lower affinity to sTnC but still induces widespread perturbations in both domains. Extensive spectral overlap precludes definite assignment of intermolecular nuclear Overhauser effect (NOEs) although intraprotein NOEs do indicate the nature of some drug-induced conformational changes. Relaxation enhancements induced by two spin-labeled calmidazolium analogues demonstrate that several methionine residues of CaM, significantly immobilized by calmidazolium binding, are in fact located at or near its binding sites. These and other residue-specific broadening effects have enabled low resolution models to be constructed of the predominantly hydrophobic drug-binding sites on each domain of CaM. The hydrophobic portions of calmidazolium itself, and its analogues, contact side chains of Ala-15, Leu-18, Phe-19, Val-35, Met-36, Leu-37, Leu-39, Met-51, Met-71, Met-72, and Met-76 in the N-terminal domain of calmodulin, and Ala-88, Val-91, Phe-92, Val-108, Met-109, Leu-112, Phe-141, and Met-145 in its C-terminal domain. The model, and an analogous one of sTnC, can be used to rationalize drug-induced changes in intraprotein NOEs. Issues pertaining to the possible simultaneous binding of calmidazolium to both globular domains of the proteins are discussed in terms of the experimental results and the overall structures of each protein.

A proton nuclear magnetic resonance and molecular modeling study of cardiac troponin C. Calcium dependence and aromatic spectral assignments.

Proton (1H) NMR at 360 MHz has been used to characterize calcium-induced spectral changes in bovine cardiac troponin C in more detail than hitherto reported (Hincke, M. T., Sykes, B. D., and Kay, C. M. (1981) Biochemistry 20, 3286-3294). The observed changes are consistent with two equivalents of calcium occupying high affinity sites, with subsequent binding of a single equivalent to a lower affinity site. Two-dimensional J-correlated and nuclear Overhauser effect NOE-correlated and conventional one-dimensional NOE experiments, combined with a consideration of the titration behavior, have allowed all the aromatic signals, and several prominently shifted alpha-CH and methyl group signals, as well as some methionine methyl signals of the calcium-saturated protein, to be assigned. This exercise was facilitated by the construction of a model of the calcium-bound protein based on crystal structure data of the homologous calmodulin and skeletal troponin C, using mutations, energy minimizations, and molecular dynamics simulations, combined with the ring-current shift and NOE prediction program PARSNIP (Reid, D. G., and Saunders, M. R. (1989) J. Biol. Chem. 264, 2003-2012).

Binding of a calcium sensitizer, bepridil, to cardiac troponin C. A fluorescence stopped-flow kinetic, circular dichroism, and proton nuclear magnetic resonance study.

Stopped-flow fluorescence kinetic measurements, circular dichroism (CD), and 1H nuclear magnetic resonance (NMR) spectroscopy at 360 MHz have been used to study the interaction of the calcium-channel blocker and calmodulin antagonist bepridil with cardiac troponin C (cTnC) in the presence of calcium. The kinetic data show that bepridil reduces the rate of calcium release only from the low affinity, calcium-specific site and not from the two high affinity calcium/magnesium sites. CD measurements indicate that drug binding leads to a small increase in the alpha-helical content of the complex. 1H NMR shows that the protein binds one equivalent of bepridil, with a dissociation constant of approximately 20 microM, only when the low affinity calcium site is occupied. Exchange is fast or intermediate on the chemical shift time scale. Drug binding is shown to be largely localized in the N-terminal domain, containing the low affinity calcium site, by observing the shifting and broadening of several resonances associated with that domain. These include assigned aromatic signals together with methionyl and other methyl signals. Observation of intermolecular nuclear Overhauser effects was precluded by extensive spectral overlap. Consideration of the data from the three techniques permitted a model of the bepridil-cTnC complex to be constructed, using the model of cTnC derived from the x-ray structure of calmodulin (MacLachlan L. K., Reid, D. G., and Carter, N. (1990) J. Biol. Chem. 265, 9754-9763). Binding of bepridil to a prominent hydrophobic depression in the N-terminal domain can be invoked to explain many of the induced changes in the spectral and kinetic properties of the protein. The implications of the model for the calcium sensitizing action of bepridil are discussed.

Calmodulin discriminates between the two enantiomers of the receptor-operated calcium channel blocker SK&F 96365: a study using 1H-NMR and chiral HPLC.

1H nuclear magnetic resonance at 360 MHz shows that SK&F 96365 (1-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H- imidazole hydrochloride), an antagonist of mammalian receptor-operated calcium channels, interacts with the calcium-binding regulatory protein calmodulin (CaM). This may be inferred by a number of chemical shift changes in the spectrum of the calcium-saturated protein induced by addition of the compound. Moreover, two well-resolved singlets corresponding to the 2-proton of the SK&F 96365 imidazolium moiety are observed in the spectrum over a wide range of protein:compound ratios. Separation of rac SK&F 96365 into its two enantiomers by high-performance liquid chromatography on a cellulose tris (4-methylbenzoate) column enabled us to show that the doubling of this NMR signal in the presence of CaM is due to a propensity of the protein to distinguish between the two optical isomers of the compound.

Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.

A proton and fluorine-19 nuclear magnetic resonance and fluorescence study of the binding of some natural and synthetic thyromimetics to prealbumin (transthyretin).

Interactions between prealbumin and several thyromimetic compounds have been examined by proton magnetic resonance spectroscopy. One equivalent of thyroxine (T4) or reverse triiodothyronine (rT3) selectively broadens a number of protein signals, while addition of a second equivalent induces much less widespread changes. One equivalent of triiodothyronine (T3), however, produces much less dramatic changes, and effects comparable with T4 and rT3 are only apparent when a second equivalent binds. The broadening is ascribed to immobilization of flexible residues. The non-halogenated analogue 3,5-dimethyl-3'-isopropylthyronine induces qualitatively different changes suggesting incomplete entry into the thyromimetic binding channel. The fluorinated analogue SK&F 95049 (3,5-bis-thiotrifluoromethyl-3'-isopropylthyronine) induces very similar changes to T3. A fluorine-19 NMR signal with a half-height line width of approximately 150 Hz can be observed from the bound ligand. Finally, a spin-labeled T4 analogue, with a nitroxyl on the alanyl moiety, induces changes identical to those induced by T4 itself, and additionally broadens some signals corresponding to residues at the opening of the ligand binding channel. The natural tryptophan fluorescence of the protein is shown to be a sensitive indicator of binding. The possible influence of the dynamic restrictions induced by binding the first molecule of T4 or rT3 on the protein's affinity for a second hormone is discussed. It is suggested that the first interaction confers rigidity on the second site and reduces its ability to flex open and accommodate a second thyromimetic, which results in the marked negative co-operativity associated with the occupancy of this site.