Replacement of Tyrosines by Unnatural Amino Acid Aminophenylalanine Leads to Metal-Mediated Aniline Free Radical Formation in a Copper Amine Oxidase.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu2+ center must undergo formal reduction to Cu1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast (Hansenula polymorpha) amine oxidase (HPAO) that has undergone site-specific insertion of a para-aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and pKa, we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

Distraction by steady-state sounds: Evidence for a graded attentional model of auditory distraction.

Sound disrupts short-term retention in working memory even when the sound is completely irrelevant and has to be ignored. The dominant view in the literature is that this type of disruption is essentially limited to so-called changing-state distractor sequences with acoustic changes between successive distractor objects (e.g., "ABABABAB") and does not occur with so-called steady-state distractor sequences that are composed of a single repeated distractor object (e.g., "AAAAAAAA"). Here we show that this view can no longer be maintained. What is more, disruption by steady-state distractors is significantly reduced after preexposure to the distractor item, directly confirming a central assumption of attentional explanations of auditory distraction and parallel to what has been shown earlier for changing-state sounds. Taken together, the findings reported here are compatible with a graded attentional account of auditory disruption, and they are incompatible with the duplex-mechanism account. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Reassessing the token set size effect on serial recall: Implications for theories of auditory distraction.

Sequences of auditory objects such as one-syllable words or brief sounds disrupt serial recall of visually presented targets even when the auditory objects are completely irrelevant for the task at hand. The token set size effect is a label for the claim that disruption increases only when moving from a 1-token distractor sequence (e.g., "AAAAAAAA") to a token set size of 2 (e.g., "ABABABAB") but remains constant when moving from a token set size of 2 to a larger token set size (e.g., "ABCABCAB" or "DAGCFBEH"). Here we show that this claim was incorrect and based on experiments with insufficient statistical power. With sufficient statistical power it can be shown that disruption increases not only when the distractor token set size increases from 1 to 2, but also when it increases from two to eight one-syllable words (Experiment 1) and brief instrumental sounds (Experiment 2). These findings have implications for theories of auditory distraction which differ in their predictions about whether the distractor-induced performance decrement should (a) only be determined by acoustic differences between immediately adjacent distractor tokens (duplex-mechanism account) or (b) gradually increase as a function of the variability in the distractor set (attentional account). The present data are inconsistent with the duplex-mechanism account and support the attentional account. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The impact of reference tones on the adjustment of interaural cues.

In the time-intensity trading paradigm, trading ratios are inconsistent in that they differ as a function of which cue is to be adjusted by the listener. Two explanations have been offered: First, the regression model assumes a regression to the interaural parameters of a reference tone played in alternation with the test tone to cause the phenomenon of inconsistent trading ratios. The second explanation is based on an inflated perceptual weighting of the to-be-adjusted cue. The perceptual-weight explanation has been supported by experimental results showing that the phenomenon of inconsistent trading ratios appears even in the absence of a reference tone. Those findings render regression as the sole explanation for inconsistent trading ratios implausible. The experiments presented in this paper address the question whether regression to the parameters of a reference tone plays a role if a reference tone is presented. Three experiments were conducted in which trials with and without reference tone were compared directly. Both within- and between-subject comparisons showed that a reference tone affects trading ratios and location judgments if present. Although regression cannot be the sole explanation for the phenomenon of inconsistent trading ratios it seems to play a role if a reference tone is presented.

The impact of practice on the adjustment of interaural cues in a lateralization task.

Lang and Buchner [J. Acoust. Soc. Am. 124, 3120-3131 (2008)] conducted trading experiments showing that the phenomenon of different trading ratios depending on which cue is adjusted by the listener is independent of the presentation of an explicit reference tone. Four experiments were conducted to test whether implicit reference tones during the preceding practice phase may have caused the different trading ratios. The results of Lang and Buchner were replicated, showing that an implicit reference learned during the practice phase cannot account for different trading ratios in experiments without the presentation of a reference tone.

Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses.

G*Power is a free power analysis program for a variety of statistical tests. We present extensions and improvements of the version introduced by Faul, Erdfelder, Lang, and Buchner (2007) in the domain of correlation and regression analyses. In the new version, we have added procedures to analyze the power of tests based on (1) single-sample tetrachoric correlations, (2) comparisons of dependent correlations, (3) bivariate linear regression, (4) multiple linear regression based on the random predictor model, (5) logistic regression, and (6) Poisson regression. We describe these new features and provide a brief introduction to their scope and handling.

Relative influence of interaural time and intensity differences on lateralization is modulated by attention to one or the other cue: 500-Hz sine tones.

When interaural time differences and interaural intensity differences are set into opposition, the measured trading ratio depends on which cue is adjusted by the listener. In an earlier article [Lang, A.-G., and Buchner, A., J. Acoust. Soc. Am. 124, 3120-3131 (2008)], four experiments showed that the perceived localization of a broad band sound for which differences in one cue were compensated by differences in the other cue such that the sound seemed to originate from a central position shifted back toward the location from which the sound appeared to originate before the adjustment. It was argued that attention shifted toward the effect of the to-be-adjusted cue during the compensation task, leading to an increased weighting of the to-be-adjusted cue. The use of broadband stimuli raises the question whether the "shift-back effect" was caused by attentional shifts to the effect of the to-be-adjusted binaural cue or by attention shifts to the particular frequency range which is most important for localizations based on the to-be-adjusted cue. Two experiments are reported in which sine tones of 500 Hz were used instead of broadband sounds. The shift-back effect could still be observed, supporting our original hypothesis. A control experiment showed that participants had accurate representations of the critical central position.

Relative influence of interaural time and intensity differences on lateralization is modulated by attention to one or the other cue.

When setting interaural time differences and interaural intensity differences into opposition the measured trading ratio depends on which of the cues is adjusted by the listener. This paper provides some evidence that the different trading ratios may be an effect of a shift of attention toward the to-be-adjusted cue. The experiments consisted of two phases. In the compensation phase, participants canceled out the effect of one preset binaural cue by adjusting a compensatory value of the other cue until the sound was located in the center. In the localization phase participants assessed the virtual location of the sounds, again using the preset values of the fixed cue, but using the values of the other cue as previously adjusted. The sounds were no longer perceived as originating from the center. Instead, their perceived location was shifted back toward the location from which they appeared to originate before the adjustment. These findings suggest that during the compensation task the to-be-adjusted sound localization cue received an increased weight compared to the other cue. We propose shifts of attention between the cues as a mechanism that could account for this finding.

G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences.

G*Power (Erdfelder, Faul, & Buchner, 1996) was designed as a general stand-alone power analysis program for statistical tests commonly used in social and behavioral research. G*Power 3 is a major extension of, and improvement over, the previous versions. It runs on widely used computer platforms (i.e., Windows XP, Windows Vista, and Mac OS X 10.4) and covers many different statistical tests of the t, F, and chi2 test families. In addition, it includes power analyses for z tests and some exact tests. G*Power 3 provides improved effect size calculators and graphic options, supports both distribution-based and design-based input modes, and offers all types of power analyses in which users might be interested. Like its predecessors, G*Power 3 is free.

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae.

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.

Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase.

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.

Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with gamma-glutamylcysteine during catalysis: stoichiometric and site-directed mutagenic analysis of arabidopsis thaliana PCS1-catalyzed phytochelatin synthesis.

Phytochelatin (PC) synthase has been assumed to be a gamma-glutamylcysteine dipeptidyl transpeptidase (EC 2.3.2.15) and, more recently, as exemplified by analyses of the immunopurified recombinant enzyme from Arabidopsis thaliana (AtPCS1-FLAG), has been shown to catalyze a PC synthetic reaction with kinetics that approximates a bisubstrate-substituted enzyme mechanism in which millimolar concentrations of free GSH and micromolar concentrations of heavy metal.GSH thiolates (e.g. cadmium.GS(2)) or millimolar concentrations of S-alkylglutathiones serve as cosubstrates. Here, we show, by direct analyses of the stoichiometry of AtPCS1-FLAG-catalyzed PC synthesis, the kinetics and stoichiometry of acylation of the enzyme and release of free glycine from gamma-Glu-Cys donors, and the effects of the Cys-to-Ser or -Ala and Ser-to-Ala substitution of conserved residues in the catalytic N-terminal half of the enzyme, that PC synthase is indeed a dipeptidyltransferase that undergoes gamma-Glu-Cys acylation at two sites during catalysis, one of which, in accord with a cysteine protease model, likely corresponds to or is at least tightly coupled with Cys(56). The identity of the second site of enzyme modification remains to be determined, but it is distinguishable from the first Cys(56)-dependent site, which is amenable to gamma-Glu-Cys acylation by free GSH, because its acylation not only depends on the provision of Cd(2+) or GSH with a blocked, S-alkylated thiol group, but is also necessary for net PC synthesis. We conclude that des-Gly-PCs are not generated as an immediate by-product, but rather that the enzyme catalyzes a dipeptidyl transfer reaction in which some of the energy liberated upon cleavage of the Cys-Gly bonds of the gamma-Glu-Cys donors in the first phase of the catalytic cycle is conserved through the formation of a two site-substituted gamma-Glu-Cys acyl-enzyme intermediate whose hydrolysis provides the energy required for the formation of the new peptide bond required for the extension of PC chain length by one gamma-Glu-Cys repeat per catalytic cycle.