NMR assignments and secondary structure distribution of emfourin, a novel proteinaceous protease inhibitor.

Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of 1H, 13C, and 15N resonances of recombinant M4in and its structural-dynamic properties derived from the chemical shifts. According the NMR data analysis, the M4in molecule comprises 3-5 helical elements and 4-6 ß-strands, at least two of which are apparently antiparallel, ascribing this obviously globular protein to the α + ß structural class. Besides, two disordered regions also exist in the central loops between the regular secondary structural elements. The obtained data provide the basis for determining the high-resolution structure as well as functioning mechanism of M4in that can be used for development of new antibacterial therapeutic strategies.

Ionization of organic molecules with metal ions formed in the laser plasma.

A laser plasma ion source was used to ionize volatile organic compounds in a gas sample. The plasma was generated on a metal target in the intermediate vacuum region of ~0.3 Torr using a pulsed Nd:YAG laser with a wavelength of 1 µm. The resulting ions mass spectra were acquired using orthogonal time-of-flight mass spectrometer (O-TOF MS). When using a copper target, the ions formed are simple complexes (CuM+ ) of copper ions with organic molecules. The possibility of online identification of trace amounts of alkanes in nitrogen and air, with a detection limit of ~10 ppb, was demonstrated. The ionization efficiency of volatile organic compounds through the formation of clusters with metal ions is 10-4 in terms of the quasimolecular complex ions. The rate constants of ion-molecular reactions of copper ions with octane and water molecules in nitrogen and air are estimated.

The protealysin operon encodes emfourin, a prototype of a novel family of protein metalloprotease inhibitors.

Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (Ki = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases.

Numerical simulation of ion transport in an atmosphere-to-vacuum interface taking into account gas dynamics and space charge.

A two-step approach was developed for the study of ion transport in an atmospheric pressure interface. In the first step, the flow in the interface was numerically simulated using the standard gas dynamic package ANSYS CFX 15.0. In the second step, the calculated fields of pressure, temperature, and velocity were imported into a custom-built software application for simulation of ion motion under the influence of both gas dynamic and electrostatic forces. To account for space charge effects in axially symmetric interfaces an analytical expression was used for the Coulomb force. For all other types of interfaces, an iterative approach for the Coulomb force computation was developed. The simulations show that the influence of the space charge is the main contributor to the loss of ion current in the heated capillary. In addition, the maximum ion current which can be transmitted through the heated capillary (0.58 mm inner diameter and 58.5 mm length) is limited to ∼6 nA for ions with m/z = 508 Da and with reduced ion mobility 1.05 cm2V-1s-1. This limit remains practically constant and independent of the ion current at the entrance of the capillary. For a particular ion type, this limit depends on its m/z ratio and ion mobility.

Processing of protealysin precursor.

Protealysin, a protease previously described by us in Serratia proteamaculans, belongs to the group of thermolysin-like proteases (TLPs) that differ from classical TLPs by the precursor structural organization. The propeptide of protealysin precursor has no significant structural similarity to the propeptides of most TLPs. The functions of protealysin-like precursors and mechanisms of their action remain unclear. We studied the pathway of protealysin precursor processing in vitro using standard approaches: modification of the catalytic site and monitoring immobilized precursor maturation. The Glu(113) --> Ala substitution inhibited the precursor maturation, which pointed to the autocatalytic processing. The mutant precursor exposure to active protealysin converted it to the mature enzyme, thus, indicating the intermolecular processing. Intermolecular processing of the mutant protein by other proteases such as thermolysin or subtilisin is also possible. The intact protealysin precursor was efficiently autoprocessed in solution but not after immobilization. These data indicate that the processing of protealysin precursor differs from that of classical TLPs. The protealysin propeptide is cleaved by an autocatalytic or heterocatalytic intermolecular mechanism and is most likely not removed intramolecularly.

Chlorofullerene C60Cl6: a precursor for straightforward preparation of highly water-soluble polycarboxylic fullerene derivatives active against HIV.

We report for the first time the application of chlorofullerene C60Cl6 as a substrate for straightforward preparation of highly water-soluble fullerene derivatives, promising compounds for investigation of the biological action of fullerenes in vitro and in vivo. Methyl esters of phenylacetic and benzylmalonic acids were used as reagents in the Friedel-Crafts arylation of C60Cl6 that resulted in the corresponding C60(Ar)5Cl compounds with 50-60% yields. The following cleavage of ester groups in phenylacetic and benzylmalonic residues was accomplished almost quantitatively to yield the corresponding fullerene-based acids bearing 5 and 10 carboxylic groups, respectively. The relatively-low solubility of these acids in water can be strongly enhanced (up to 150-200 mg ml(-1)) by their conversion to salts with alkali metal cations. These fullerene salt derivatives showed pronounced anti-HIV action and low toxicity; these two findings point to the necessity for further investigation of the biological properties of the here-reported compounds.