A Blind Test of Computational Technique for Predicting the Likelihood of Peptide Sequences to Cyclize

An in silico computational technique for predicting peptide sequences that can be cyclized by cyanobactin macrocyclases, e.g., PatGmac, is reported. We demonstrate that the propensity for PatGmac-mediated cyclization correlates strongly with the free energy of the so-called pre-cyclization conformation (PCC), which is a fold where the cyclizing sequence C and N termini are in close proximity. This conclusion is driven by comparison of the predictions of boxed molecular dynamics (BXD) with experimental data, which have achieved an accuracy of 84%. A true blind test rather than training of the model is reported here as the in silico tool was developed before any experimental data was given, and no parameters of computations were adjusted to fit the data. The success of the blind test provides fundamental understanding of the molecular mechanism of cyclization by cyanobactin macrocyclases, suggesting that formation of PCC is the rate-determining step. PCC formation might also play a part in other processes of cyclic peptides production and on the practical side the suggested tool might become useful for finding cyclizable peptide sequences in general

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content

Možnosti a způsoby využití kalů a sedimentů z ČOV

Stav nakládání s čistírenskými kaly v ČR - aktualizace za období 2002-2004(5) s vazbou na trendy a vývoj legislativy. Vývoj produkce kalů z komunálních ČOV a nakládání s kaly. Charakteristika technického stavu kalového hospodářství v ČR. Hlavní provozně využitelné metody hygienizace kalu. Provozní zkušenosti s přímou aplikací kalů na zemědělskou půdu. Vliv technologie odstranění nutrientů z odpadních vod na produkci kalů. Prognóza vývoje následného využití/odstranění kalu z ČOV. Dosahované složení kalů a limity rizikových látek v kalech ve vztahu k platné legislativě. Nové trendy nakládání s kaly

The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain

Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications