Amphitrite: a program for processing travelling wave ion mobility mass spectrometry data

Since the introduction of travelling wave (T-Wave)-based ion mobility in 2007 a large number of research laboratories have embraced the technique, particularly those working in the field of structural biology. The development of software to process the data generated from this technique, however, has been limited. We present a novel software package that enables the processing of T-Wave ion mobility data. The program can deconvolute components in a mass spectrum and uses this information to extract corresponding arrival time distributions (ATDs) with minimal user intervention. It can also be used to automatically create a collision cross section (CCS) calibration and apply this to subsequent files of interest. A number of applications of the software, and how it enhances the information content extracted from the raw data, are illustrated using model proteins

The respiratory arsenite oxidase:structure and the role of residues surrounding the rieske cluster

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large alpha subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small beta subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a -20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc(1) complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc(1) complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter

An analysis of B-cell epitope discontinuity

Although it is widely acknowledged that most B-cell epitopes are discontinuous, the degree of discontinuity is poorly understood. For example, given that an antigen having a single epitope that has been chopped into peptides of a specific length, what is the likelihood that one of the peptides will span all the residues belonging to that epitope? Or, alternatively, what is the largest proportion of the epitope's residues that any peptide is likely to contain? These and similar questions are of direct relevance both to computational methods that aim to predict the location of epitopes from sequence (linear B-cell epitope prediction methods) and window-based experimental methods that aim to locate epitopes by assessing the strength of antibody binding to synthetic peptides on a chip. In this paper we present an analysis of the degree of B-cell epitope discontinuity, both in terms of the structural epitopes defined by a set of antigen–antibody complexes in the Protein Data Bank, and with respect to the distribution of key residues that form functional epitopes. We show that, taking a strict definition of discontinuity, all the epitopes in our data set are discontinuous. More significantly, we provide explicit guidance about the choice of peptide length when using window-based B-cell epitope prediction and mapping techniques based on a detailed analysis of the likely effectiveness of different lengths

EPR properties of the WT and mutated Aio Rieske centres from NT-26.

<p>All spectra were recorded during titration on entirely reduced isolated complex under non-saturating conditions. Instrument settings: microwave frequency, 9,48 GHz; modulation amplitude 1.6 mT, temperature 15 K; microwave power, 6.3 mT.</p

Comparison of arsenite oxidase activities in total cell extracts of <i>E. coli</i> strains.

<p>DH5α and JM109λ<i>pir</i> grown were grown with oxygen, nitrate and DMSO as terminal electron acceptors. Error bars represent the average of six individual experiments.</p

Close up view of the Aio Rieske 2Fe-2S cluster.

<p>A wall eye stereo superposition of NT-26 (yellow cartoon, with sticks having yellow coloured carbon atoms), <i>A. faecalis</i> (teal cartoon, with sticks having white coloured carbon atoms) and <i>R. sphaeroides</i> (2qjk) <i>bc</i>₁ complex (salmon cartoon, with sticks having cyan coloured carbon atoms). Nitrogen atoms are coloured blue, oxygen atoms coloured red and sulphur atoms green when shown in stick in all structures. The Rieske cluster from the NT-26 structure is shown with iron atoms as brown spheres and sulphur atoms as dull yellow spheres. The residues in the NT-26 are labelled and the corresponding atoms in the other structures are discussed in the text. The superposition was generated by using all backbone atoms from residue 104 to residue 110 in the NT-26 Aio structure as the template. This provides a more meaningful view of changes at the Rieske cluster than a simple all atom superposition.</p

The heterodimeric structure of the Aio from NT-26.

<p>The Aio consists of an α (pale blue) and β chain (pale yellow). The pterin co-factor, 3Fe-4S, 2Fe-2S clusters are shown as space filling spheres. Residues which ligate the clusters are shown as sticks, as are the two residues surrounding the Rieske cluster (K106 and F108). Atoms are coloured iron orange, sulphur dark yellow, carbon bright yellow, molybdenum green, phosphorus bright orange, oxygen red, nitrogen blue.</p

Purification table of NT-26 recombinant arsenite oxidase.

<p>Purification table of NT-26 recombinant arsenite oxidase.</p