A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid

Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+? termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.     

New Tests to Measure Individual Differences in Matching and Labelling Facial Expressions of Emotion,and Their Association with Ability to Recognise Vocal Emotions and Facial Identity

Although good tests are available for diagnosing clinical impairments in face expression processing, there is a lack of strong tests for assessing “individual differences” – that is, differences in ability between individuals within the typical, nonclinical, range. Here, we develop two new tests, one for expression perception (an odd-man-out matching task in which participants select which one of three faces displays a different expression) and one additionally requiring explicit identification of the emotion (a labelling task in which participants select one of six verbal labels). We demonstrate validity (careful check of individual items, large inversion effects, independence from nonverbal IQ, convergent validity with a previous labelling task), reliability (Cronbach’s alphas of.77 and.76 respectively), and wide individual differences across the typical population. We then demonstrate the usefulness of the tests by addressing theoretical questions regarding the structure of face processing, specifically the extent to which the following processes are common or distinct: (a) perceptual matching and explicit labelling of expression (modest correlation between matching and labelling supported partial independence); (b) judgement of expressions from faces and voices (results argued labelling tasks tap into a multi-modal system, while matching tasks tap distinct perceptual processes); and (c) expression and identity processing (results argued for a common first step of perceptual processing for expression and identity).     

Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.     

Syntheses of novel azasugar-containing mimics of heparan sulfate fragments as potential heparanase inhibitors

A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.     

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.