A Modular Bioplatform Based on a Versatile Supramolecular Multienzyme Complex Directly Attached to Graphene

© 2016 American Chemical Society. Developing generic strategies for building adaptable or multifunctional bioplatforms is challenging, in particular because protein immobilization onto surfaces often causes loss of protein function and because multifunctionality usually necessitates specific combinations of heterogeneous elements. Here, we introduce a generic, modular bioplatform construction strategy that uses cage-like supramolecular multienzyme complexes as highly adaptable building blocks immobilized directly and noncovalently on graphene. Thermoplasma acidophilum dihydrolipoyl acyltransferase (E2) supramolecular complexes organize as a monolayer or can be controllably transferred onto graphene, preserving their supramolecular form with specific molecular recognition capability and capacity for engineering multifunctionality. This E2-graphene platform can bind enzymes (here, E1, E2's physiological partner) without loss of enzyme function; in this test case, E1 catalytic activity was detected on E2-graphene over 6 orders of magnitude in substrate concentration. The E2-graphene platform can be multiplexed via patterned cotransfer of differently modified E2 complexes. As the E2 complexes are robust and highly customizable, E2-graphene is a platform onto which multiple functionalities can be built

Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by lipoate protein ligase (LplA) or by lipoic acid synthetase (LipA) and lipoyl(octanoyl) transferase (LipB) combined. Whereas bacterial and eukaryotic LplAs comprise a single, two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The structure of Thermoplasma acidophilum LplA-N is known, but the structure of LplA-C and its role in lipoylation are unknown. We have determined the structures of the substrate-free LplA-N+LplA-C complex and the dihydrolipoyl acyltransferase lipoyl domain (E2lipD) that is lipoylated by LplA-N+LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: LplA-C is disordered but folds upon association with LplA-N; LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; the adenylate binding region of LplA-N+LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; LplA-N+LplA-C and E2lipD do not interact in the absence of substrate; LplA-N+LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; LplA-N+LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.<br/

Case study : using H5P to design and deliver interactive laboratory practicals

We describe the use of HTML5P (H5P) content collaboration framework to deliver an interactive, online alternative to an assessed laboratory practical on the Biomedical Cell Biology unit at the Manchester Metropolitan University, U.K. H5P is free, open-source technology to deliver bespoke interactive, self-paced online sessions. To determine if the use of H5P affected learning and student attainment, we compared the student grades among three cohorts: the 18/19 cohort who had 'wet' laboratory classes, the 19/20 cohort who had 'wet' laboratory classes with additional video support and the 20/21 cohort who had the H5P alternative. Our analysis shows that students using the H5P were not at a disadvantage to students who had 'wet' laboratory classes with regard to assessment outcomes. Student feedback, mean grade attained and an upward trend in the number of students achieving first-class marks (≥70%), indicate H5P may enhance students' learning experience and be a valuable learning source augmenting traditional practical classes in the future

A Modular Bio-Platform Based on a Versatile Supramolecular Multi-Enzyme Complex Directly Attached to Graphene

Developing generic strategies for building adaptable or multi-functional bio-platforms is challenging, in particular because protein immobilization onto surfaces often causes loss of protein function and multi-functionality usually necessitates specific combinations of heterogeneous elements. Here we introduce a generic, modular bio-platform construction strategy that uses cage-like supramolecular multi-enzyme complexes as highly adaptable building blocks immobilized directly and non-covalently on graphene. Thermoplasma acidophilum dihydrolipoyl acyltransferase (E2) supramolecular complexes organize as a monolayer or can be controllably transferred onto graphene, preserving their supramolecular form with specific molecular recognition capability and capacity for engineering multi-functionality. This E2-graphene platform can bind enzymes (here, E1, E2’s physiological partner) without loss of enzyme function; in this test case, E1 catalytic activity was detected on E2-graphene over six orders of magnitude in substrate concentration. The E2-graphene platform can be multiplexed via patterned co-transfer of differently modified E2 complexes. As the E2 complexes are robust and highly customizable, E2-graphene is a platform onto which multiple functionalities can be built

The benefits of using case study focussed, problem based learning approaches to unit design for Biomedical Science students

As part of the Biomedical Sciences undergraduate degree course students are required to apply biological principles to the interpretation of clinical case studies and the diagnosis of patients. Case study-based learning i.e., application of knowledge to patient diagnosis, is new to most students as case studies do not form part of non-applied A level courses in biological sciences. This approach is an example of Problem Based Learning (PBL) which has been shown to support higher levels of student learning, encouraging critical thinking and analysis. PBL approaches have also been shown to increase academic satisfaction and student engagement. In recent years we have observed a downwards trend instudent engagement and historically student performance in applied case study-based assessments to be lower than that observed for assessments based on detailing fundamental biological principles. We hypothesised that PBL teaching delivery would support students in preparinge for case study-based assessments, helping them todemonstrate their critical evaluation and problem-solving skills, and hence, improve student performance. We also hypothesised that the student learning experience would be enhanced by a PBL teaching delivery approach which would improve overall engagement. We therefore redesigned a second year Biomedical Sciences degree haematology and clinical biochemistry unit: ‘Blood Science’, with a stronger focus on PBL, including case study focussed activities throughout the unit. We subsequently analysed whether this PBL- focussed unit design improved student experience and feedback, student engagement and student confidence for biomedical science undergraduate students. We present here, our teaching strategy and the impact our changes had on student feedback for the 21/22 and 22/23 academic years. Our findings demonstrate that case study-based activities and tutorial PBL exercises, when incorporated into the curriculum design, can improve student experience in the Biomedical Sciences and other biological science undergraduate degree courses

Phylogenetic analysis of the biotin-lipoate A/B protein ligase family.

<p>Neighbor-joining phylogenetic tree of the cofactor transferase domain (Pfam03099) that includes 11,826 biotin and lipoate-ligase proteins and octanoyl-carrier proteins from eukaryotes, eubacteria and archaea. Annotation of the five broad protein domain clades as LipM (red), LplA (orange), LipL (blue), LipB (green) and biotin protein-ligase (black) clades was based upon the presence of biochemically characterized proteins within each protein set. In total, 295 archaeal sequences were included in this analysis with 161 residing in the biotin-ligase clade (54.5%) and the remainder residing in the LipM, LplA or LipB clades.</p

Phylogenetic analysis of LipM and LplA.

<p>Maximum likelihood phylogenetic tree including 131 LipM and LplA sequences from archaea, LplA sequences from major eukaryotic species (<i>S. cerevisiae, D. melanogaster</i>, <i>M. musculus</i> and <i>H. sapiens</i>) and LplA and LipM sequences from eubacteria representing Actinobacteria (<i>S. coelicolor),</i> Bacteroidetes (<i>B. thetaiotaomicron</i>), Firmicutes (<i>B. subtilis</i> and <i>S. aureus</i>) and Proteobacteria (<i>E. coli</i> and <i>B. pseudomallei).</i> Putative cases of horizontal gene transfer are indicated (asterisk) and major phylogenetic clades are highlighted: archaeal LplA (Clade I - orange; Clade II - green), LipM (red), and eukaryotic and eubacterial LplA (blue). The full phylogenetic tree including species names and bootstrap values is provided <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087063#pone.0087063.s002" target="_blank">Figure S2</a>.</p

Comparative genomic analysis of lipoylation pathways in archaea.

<p>The genomic presence of lipoylation enzymes <i>LplA-N</i>, LipM or <i>LipB</i> and their substrate OADHC <i>E2</i> is indicated. Archaeal orders lacking lipoylation pathways are highlighted (grey shading). The broad metabolic environment of each archaeal order and the number of species analyzed are also indicated. Phylogenetic relationships are based on Brochier-Armanet <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087063#pone.0087063-BrochierArmanet1" target="_blank">[53]</a>; branch lengths are not drawn to scale.</p

Genomic co-retention of <i>LipB</i> and <i>LipM</i> lipoylation genes with OADHC <i>E2</i>.

<p>(A) The presence of the <i>LipB</i> and OADHC <i>E2</i> genes in sequenced Thermoproteales genomes is indicated (species lacking both are highlighted in grey). <i>Pyrobaculum sp 1860</i> and <i>Pyrobaculum oguniense</i> have yet to be incorporated into the <i>Pyrobaculum</i> phylogeny and have been placed arbitrarily in the <i>Pyrobaculum</i> genus. (B) The presence of <i>LipM</i> and OADHC <i>E2</i> in sequenced Halobacteriales genomes is indicated (species lacking both are highlighted in grey). Phylogenetic relationships are based on Brochier-Armanet <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087063#pone.0087063-BrochierArmanet1" target="_blank">[53]</a>; branch lengths are not drawn to scale.</p