Energetics of a protein disorder–order transition in small molecule recognition

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered ‘lid’ region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry.

Molecular simulations and biophysical measurements elucidate why the ligand AM-7209 orders a disordered region of the protein MDM2 on binding. This work expands strategies available to medicinal chemists for targeting disordered proteins.     

Comparative Study of Sample Carriers for the Identification of Volatile Compounds in Biological Fluids Using Raman Spectroscopy

Vibrational spectroscopic techniques and especially Raman spectroscopy are gaining ground in substituting the officially established chromatographic methods in the identification of ethanol and other volatile substances in body fluids, such as blood, urine, saliva, semen, and vaginal fluids. Although a couple of different carriers and substrates have been employed for the biochemical analysis of these samples, most of them are suffering from important weaknesses as far as the analysis of volatile compounds is concerned. For this reason, in this study three carriers are proposed, and the respective sample preparation methods are described for the determination of ethanol in human urine samples. More specifically, a droplet of the sample on a highly reflective carrier of gold layer, a commercially available cuvette with a mirror to enhance backscattered radiation sealed with a lid, and a home designed microscope slide with a cavity coated with gold layer and covered with transparent cling film have been evaluated. Among the three proposed carriers, the last one achieved a quick, simple, and inexpensive identification of ethanol, which was used as a case study for the volatile compound, in the biological samples. The limit of detection (LoD) was found to be 1.00 μL/mL, while at the same time evaporation of ethanol was prevented.     

Organosulphur compounds-XXXVIII Axial preference of a 2-dimethoxyphosphoryl substituent in 1,3,5-trithiane

¹H-NMR studies and X-ray analysis indicate that 2-dimethoxyphosphoryl-1,3,5-trithiane exists both in solution and in the crystal in a chair conformation with the dimethoxyphosphoryl group at C-2 being axial.     

Synthesis and structure of tetracoordinated nickel(II) complexes of deprotonated chelated aminoazo ligands: X-ray crystal structure of bis[1-isopropyl-3-methyl-4-(4-methylphenylazo)-5-(4-methoxyphenylamidato)pyrazole] nickel(II)

Summary Tetracoordinate nickel(II) complexes NiL₂ derived from the deprotonated forms of aminoazoligands HL were prepared and investigated by spectroscopy and magnetic susceptibility measurements. The magnetic moments, which lie in the 3.1–3.6 B.M. range correspond to the occurrence both in solution and in the solid state of the high spin (S = 1) form and a tetrahedral configuration at the metal centre. The u.v. spectra exhibit three ligand field bands at 1020–1280 nm characteristic of high spin nickel(II) complexes. The large isotropic chemical shifts found in the ¹H n.m.r. spectra are consistent with partial delocalization of unpaired electron spin density to the ligand HOMO. The X-ray single crystal structure of NiL ₂ ⁴ [L⁴= 1-isopropyl-3-methyl-4-(4-methylphenylazo)-5-(4-methoxyphenylamino)-pyrazole] reveals that the metal is coordinated by four nitrogen atoms in tetrahedral configuration with an angle of 90° between the N(1)NiN(2) and N(6)NiN(7) planes belonging to the different almost planar metallocycles. The rates of RS interconversion of the tetrahedral configuration for NiL ₂ ⁴ and NiL ₂ ⁵ [L⁵ = 1-isopropyl-3-methyl-4-phenylazo-d ₅-5-(4-methoxyphenylamino)pyrazole] are slow on the n.m.r. timescale. In contrast to NiL ₂ ¹ -Ni ₂ ⁶ , NiL ₂ ⁷ ], which contains coordinated NH-groups instead of NAr-groups, is planar.     

Tandem mass spectrometry of multiply phosphorylated forms of a 'histidine-tag' derived from a recombinant protein kinase expressed in bacteria

When a histidine-tagged form of the protein kinase Aurora-2 was expressed in Escherichia coli, the purified product carried four to nine phosphate groups, although many fewer were expected. The amino-terminal tag had the sequence GSSHHHHHHSSGLVPRGSHMK-. Tryptic digestion of the product followed by analysis by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) showed that phosphorylation could occur on the five serine residues of the tag. Mono-, bis-, tris-, tetra- and pentaphosphorylated forms of the tag were detected, and their behavior in MS/MS was studied using a quadrupole/time-of-flight mass spectrometer. The MS/MS spectra were dominated by the products of neutral loss events (in 98 Da increments, each equivalent to loss of H3PO4), but sufficient b- and y-type sequence ions were detected to allow the locations of the phosphates to be specified in some cases. The assignment of phosphorylation sites for incompletely phosphorylated forms of the tag peptide was challenging, but it appeared that Ser-10 and Ser-11 of the tag were more likely to be phosphorylated than Ser-2 and Ser-3.     

Photodynamic inactivation of isolated crayfish mechanoreceptor neuron: different death modes under different photosensitizer concentrations

To study the mechanism of photodynamic nerve cell killing, isolated crayfish mechanoreceptor neurons were photosensitized by the sulfonated aluminum ophthalocyanine Photosens. Neuron activity was continuously recorded until irreversible abolition. Intense (10(-5) M Photosens) or weak (10(-7) M Photosens) photosensitization induced different bioelectric neuron responses: firing activation followed by irreversible depolarization block or gradual inhibition until firing abolition, respectively. These bioelectric responses were accompanied by different biochemical and morphological changes. In the case of intense photosensitization, neuron nuclei swelled and then shrank. Succinate dehydrogenase (SDH) was inhibited, and the plasma membrane was compromised just after firing cessation. Weak photosensitization did not induce these changes but caused swelling of the endoplasmic reticulum and destruction of the matrix, cristae and membranes in some of the mitochondria. Other mitochondria, however, retained the normal structure. Plasma membrane damage, SDH inhibition, nucleus shrinkage and impairment of the nuclear border occurred after 2-4 h. It is concluded that intense photosensitization induced necrotic processes during irradiation, whereas weaker impact caused delayed necrosis 2-4 h later. The observed electrophysiological neuron responses to photodynamic therapy may be considered as early hallmarks of different modes of forthcoming cell death.     

Electron-transfer kinetics and mechanism of the reduction of octacyanometallates(IV) (M = Mo,W) by hydroxide ion in aqueous solution

Summary The kinetics of the reduction of octacyanometallates(IV) in alkaline aqueous medium have been studied spectrophotometrically. The experimental results are in agreement with following rate law:-d[M(CN) _inf8 ^sup3– ]/dt = k _obs[M(CN) _inf8 ^sup3– ]²[OH^–][Na⁺] where k _obs = 4.1 × 10^–2M^–3s^–1 (Mo) and 4.0 × 10^–4 M^–3 s^–1 (W). The rate data were used to calculate the thermodynamic activation parameters H and S . A mechanism of the reaction is discussed.On leave from Faculty of Chemistry, Forest Engineering Institute, Archangelsk, Russia.     

Struktur von Gläsern des Systems TeO2−VO2

Glasses from the TeO₂–VO₂ system and - and -crystalline modifications of TeO₂·VO₂ (TeVO₄) are studied by IR-spectroscopy in the 1400–400 cm^–1 range. Similarity is established in the area near 970 cm^–1 of the spectrum between TeVO₄ glass and the -form. The continuous shift of the band from 970 cm^–1 to 950 cm^–1 with increasing TeO₂ content in the glasses is connected with the indirect influence on the nonbridging V–O bond.It is shown that the local environment of the V in some glasses from TeO₂–VO₂ and TeO₂–V₂O₅ system is similar: VO₅ polyhedra.     

Synthesen,chemische und elektrische Eigenschaften von Tetrathiafulvalen mit 1,6-Methano[10]annulen-Partialstruktur und von Chalcogendiimid-derivaten

Syntheses, Chemical and Electrical Properties of Tetrathiafulvalene with a 1,6-Methano[10]annulene Moiety and of Chalcogendiimide-Derivatives The synthesis of the tetrathiafulvalene 6 has been described: the reaction of 1 with the benzenedithoil 2 yielded the bis-dithioacetal 3 of 1,6-methano[10]annulene-dicarbaldehyde 1 . The oxidation of 3 in the presence of HBF₄ in Et₂O yielded the salt 4 , elimination of a hydride anion with triphenylmethylium tetrafluoroborate the salt 5 , and elimination of a proton in 5 with Et₃N the tetrathiafulvalene 6 with a 1,6-methano[10]annulene moiety. The spectroscopic and electrochemical properties of 6 are described, and also the syntheses and properties of charge-trasnfer complexes 7 and 8 , including the properties of electric conductivity of 7 and 8 . the syntheses of 9-13 are reported and also their spectroscopic properties.