Exploration of a potential difluoromethyl-nucleoside substrate with the fluorinase enzyme

The authors thank EPSRC and the Scottish Imaging Network (SINAPSE) for grants. DO’H thanks the Royal Society for a Wolfson Research Merit Award and ST is grateful to the John and Kathleen Watson Scholarship for financial support.The investigation of a difluoromethyl-bearing nucleoside with the fluorinase enzyme is described. 5’,5’–Difluoro-5’-deoxyadenosine 7 (F2DA) was synthesised from adenosine, and found to bind to the fluorinase enzyme by isothermal titration calorimetry with similar affinity compared to 5’–fluoro-5’-deoxyadenosine 2 (FDA), the natural product of the enzymatic reaction. F2DA 7 was found, however, not to undergo the enzyme catalysed reaction with l–selenomethionine, unlike FDA 2, which undergoes reaction with l-selenomethionine to generate Se-adenosylselenomethionine. A co-crystal structure of the fluorinase and F2DA 7 and tartrate was solved to 1.8 Å, and revealed that the difluoromethyl group bridges interactions known to be essential for activation of fluoride for reaction. An unusual hydrogen bonding interaction between the hydrogen of the difluoromethyl group and one of the hydroxyl oxygens of the tartrate ligand was also observed. The bridging interactions, coupled with the inherently stronger C–F bond in the difluoromethyl group, offers an explanation for why no reaction is observed.PostprintPeer reviewe

Sparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channels

BEB is grateful for funding from the European Union (Marie Curie Actions REA 334496). This work was supported by the EPSRC (EP/M024660/1) and the Wellcome Trust (099149/Z/12/Z). CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government.Pulse EPR is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g. homo-multimers) due to distorting multi-spin effects. Without suppression of these effects much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multi-spin problem.Here, two different appro aches for suppressing multi-spin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both, the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of E. coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multi-spin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analogue.Publisher PDFPeer reviewe

The structural biology of patellamide biosynthesis

This work was supported by grants from the ERC339367 (JHN and MJ) and BBSRCBB/K015508/1 (JHN and MJ).The biosynthetic pathways for patellamide and related natural products have recently been studied by structural biology. These pathways produce molecules that have a complex framework and exhibit a diverse array of activity due to the variability of the amino acids that are found in them. As these molecules are difficult to synthesize chemically, exploitation of their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization; closely related products are also prenylated. Enzyme activities have been identified for all these transformations except epimerization, which may be spontaneous. This review highlights the recent structural and mechanistic work on amino acid heterocyclization, peptide cleavage and peptide macrocyclization. This work should help in using the enzymes to produce novel analogs of the natural products enabling an exploitation of their properties.Peer reviewe

TarO : a target optimisation system for structural biology

This work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) Structural Proteomics of Rational Targets (SPoRT) initiative, (Grant BBS/B/14434). Funding to pay the Open Access publication charges for this article was provided by BBSRC.TarO (http://www.compbio.dundee.ac.uk/taro) offers a single point of reference for key bioinformatics analyses relevant to selecting proteins or domains for study by structural biology techniques. The protein sequence is analysed by 17 algorithms and compared to 8 databases. TarO gathers putative homologues, including orthologues, and then obtains predictions of properties for these sequences including crystallisation propensity, protein disorder and post-translational modifications. Analyses are run on a high-performance computing cluster, the results integrated, stored in a database and accessed through a web-based user interface. Output is in tabulated format and in the form of an annotated multiple sequence alignment (MSA) that may be edited interactively in the program Jalview. TarO also simplifies the gathering of additional annotations via the Distributed Annotation System, both from the MSA in Jalview and through links to Dasty2. Routes to other information gateways are included, for example to relevant pages from UniProt, COG and the Conserved Domains Database. Open access to TarO is available from a guest account with private accounts for academic use available on request. Future development of TarO will include further analysis steps and integration with the Protein Information Management System (PIMS), a sister project in the BBSRC Structural Proteomics of Rational Targets initiative.Publisher PDFPeer reviewe

Mismatch negativity/P3a complex in young people with psychiatric disorders : a cluster analysis

Background: We have recently shown that the event-related potential biomarkers, mismatch negativity (MMN) and P3a, are similarly impaired in young patients with schizophrenia- and affective-spectrum psychoses as well as those with bipolar disorder. A data driven approach may help to further elucidate novel patterns of MMN/P3a amplitudes that characterise distinct subgroups in patients with emerging psychiatric disorders. Methods: Eighty seven outpatients (16 to 30 years) were assessed: 19 diagnosed with a depressive disorder; 26 with a bipolar disorder; and 42 with a psychotic disorder. The MMN/P3a complex was elicited using a two-tone passive auditory oddball paradigm with duration deviant tones. Hierarchical cluster analysis utilising frontal, central and temporal neurophysiological variables was conducted. Results: Three clusters were determined: the 'globally impaired' cluster (n = 53) displayed reduced frontal and temporal MMN as well as reduced central P3a amplitudes; the 'largest frontal MMN' cluster (n = 17) were distinguished by increased frontal MMN amplitudes and the 'largest temporal MMN' cluster (n = 17) was characterised by increases in temporal MMN only. Notably, 55% of those in the globally impaired cluster were diagnosed with schizophrenia-spectrum disorder, whereas the three patient subgroups were equally represented in the remaining two clusters. The three cluster-groups did not differ in their current symptomatology; however, the globally impaired cluster was the most neuropsychologically impaired, compared with controls. Conclusions: These findings suggest that in emerging psychiatric disorders there are distinct MMN/P3a profiles of patient subgroups independent of current symptomatology. Schizophrenia-spectrum patients tended to show the most global impairments in this neurophysiological complex. Two other subgroups of patients were found to have neurophysiological profiles suggestive of quite different neurobiological (and hence, treatment) implications

Frontal lobe changes occur early in the course of affective disorders in young people

<p>Abstract</p> <p>Background</p> <p>More severe and persistent forms of affective disorders are accompanied by grey matter loss in key frontal and temporal structures. It is unclear whether such changes precede the onset of illness, occur early in the course or develop gradually with persistence or recurrence of illness. A total of 47 young people presenting with admixtures of depressive and psychotic symptoms were recruited from specialist early intervention services along with 33 age matched healthy control subjects. All participants underwent magnetic resonance imaging and patients were rated clinically as to current stage of illness. Twenty-three patients were identified as being at an early 'attenuated syndrome' stage, while the remaining were rated as having already reached the 'discrete disorder' or 'persistent or recurrent illness' stage. Contrasts were carried out between controls subjects and patients cohorts with attenuated syndromes and discrete disorders, separately.</p> <p>Results</p> <p>The patients that were identified as having a discrete or persisting disorder demonstrated decreased grey matter volumes within distributed frontal brain regions when contrasted to both the control subjects as well as those patients in the attenuated syndrome stage. Overall, patients who were diagnosed as more advanced in terms of the clinical stage of their illness, exhibited the greatest grey matter volume loss of all groups.</p> <p>Conclusions</p> <p>This study suggests that, in terms of frontal grey matter changes, a major transition point may occur in the course of affective illness between early attenuated syndromes and later discrete illness stages.</p

Derivatisable cyanobactin analogues : a semisynthetic approach

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1) and the Wellcome Trust (Triple TOF 5600 mass spectrometer (094476), the MALDI TOF-TOF Analyser (079272AIA), 700 NMR and supported G.M. (097831)). J.H.N. is a Royal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University.Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine-tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger-scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.Publisher PDFPeer reviewe

PELDOR in rotationally symmetric homo-oligomers

This article was made open access through BIS OA funding.Nanometre distance measurements by pulsed electron−electron double resonance (PELDOR) spectroscopy have become an increasingly important tool in structural biology. The theoretical underpinning of the experiment is well-defined for systems containing two nitroxide spin-labels (spin pairs) however recently experiments have been reported on homo-oligomeric membrane proteins consisting of up to eight spin-labelled monomers. We have explored the theory behind these systems by examining model systems based on multiple spins arranged in rotationally symmetric polygons. The results demonstrate that with a rising number of spins within the test molecule, increasingly strong distortions appear in distance distributions obtained from an analysis based on the simple spin pair approach. These distortions are significant over a range of system sizes and remain so even when random errors are introduced into the symmetry of the model. We present an alternative approach to the extraction of distances on such systems based on a minimisation that properly treats multi-spin correlations. We demonstrate the utility of this approach on a spin-labelled mutant of the heptameric Mechanosensitive Channel of Small Conductance of E. coli.Publisher PDFPeer reviewe

Taking a molecular motor for a spin : helicase mechanism studied by spin labelling and PELDOR

Welcome Trust programme grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust multi-user equipment grant [099149/Z/12/Z]. Royal Society Wolfseon Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The complex molecular motions central to the functions of helicases have long attracted attention. Protein crystallography has provided transformative insights into these dynamic conformational changes, however important questions about the true nature of helicase configurations during the catalytic cycle remain. Using pulsed EPR (PELDOR or DEER) to measure interdomain distances in solution, we have examined two representative helicases: PcrA from superfamily 1 and XPD from superfamily 2. The data show that PcrA is a dynamic structure with domain movements that correlate with particular functional states, confirming and extending the information gleaned from crystal structures and other techniques. XPD in contrast is shown to be a rigid protein with almost no conformational changes resulting from nucleotide or DNA binding, which is well described by static crystal structures. Our results highlight the complimentary nature of PELDOR to crystallography and the power of its precision in understanding the conformational changes relevant to helicase function.Publisher PDFPeer reviewe