β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural system

β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural system

Human Creativity and Invention: Exploring the stimuli of invention within the German Makers Community

Exploring the stimuli of invention within the German Makers Community ‘A piece of wood becomes, in the child’s mind, a wand. A building block becomes a mobile phone. The child plays at being the mother in the home corner,’ (Smidt, 2009, p. 105). The research on human creativity and the talent for the invention are long studied phenomena in the history of human evolution. Nowadays these two terminologies are often substituted by the notion of innovation. The work presented here, first of all, examines the question of how historically and culturally a distinction was drawn between invention and innovation. The work examines ancient German documents from the late eighteenth century, explores and defines a concept of the invention that is exempt from economic thought, and thus distinguishes the term invention from the form of innovation in use today, which was principally characterised by Joseph Alois Schumpeter in the 19th century in the age of Industrialisation. Therefore, the thesis is focusing on past, and contemporary research on the definitions of the invention to come up with a preconception for stimuli of invention. The work uses the process of the hermeneutic circle that moves from a precursory understanding of some parts to the whole the topic of human creativity and invention and a global understanding of the whole context back to an improved understanding of each part, i.e., the invention concept and the meaning of stimuli of invention. The invention concept has been developed further according to themes, in which historical and contemporary research findings on creativity, invention and innovation are incorporated. The invention concept will be placed according to time and structure, and deficiencies will be identified in present-day societal and cultural considerations because of the much expanded, economically shaped objectives. Also, a methodical approach will be used to define and apply the stimuli of invention, using the Systems Model of Creativity (SMoC) of Mihaly Csikszentmihalyi. While the systems model in its origin describes the information flow and the transformation of information between the three systems domain, field and the individual, the modification here includes the transactions of the three systems by implying factors that stimulate creativity and invention. New insights of contemporary creativity research then extend these number of stimuli of invention and the stimuli of invention are studied in the German Maker community. In general, the German Maker Community is about individuals that share their knowledge and ideas openly. Moreover, even if there is a focus on Makers living in Germany, these people are globally interconnected, and distribute and gather their knowledge from an engaged, global community supported by contemporary information technology. In addition, the context of the Maker community as an internationally recognised global movement for innovation and experimentation follows the thread to have potential people that give an insight about the stimuli motivating them to participate, contribute and share ideas, inventions and innovations in the Maker Movement, its communities, or its subcultural communities. The demarcation of the Maker Movement is still one of the open issues, therefore, the focus of the investigation was on people immersed in the ‘Community of Making’. As a result, the descriptions included in this thesis may illuminate the issues why people are deeply devoted to the invention in this community, even without remuneration or economic benefit or even of own cost. The research question, which factors facilitate inventions in the German Maker Community, is being answered. On the assumption that the German Maker Community belongs to the international Maker Movement, the study illustrates that the German Maker Community has partly other roots according to the cases investigated. Nevertheless, the objectives, practices and concerns have converged to the extent that a comparison between research results on the international Maker Movement and the German Maker Community can be drawn. Based on the comparison and the detailed investigation of distinctions, new insights into the socio-cultural structure of the Maker Community have been obtained, which also serve to explain how the Stimuli of Invention (SI) operate within the maker community. In the last section, the question of how German Makers can improve their skills to invent is discussed in more detail. On the interrelationships of the three systems Domain, Field and Individual in the Systems Model for Creativity (SMoC), additional themes are presented which deliver answers about the German Makers and possibly also for the international Maker Movement how these individuals continuously improve their skills to invent to the better for upcoming generations. ‘Ensure that you continue to offer children many opportunities to follow their passions and interests as the interaction with others, use cultural tools and solve problems,’ (Smidt, 2009, p. 116). The profound insight about learning and education in this context of the German Maker Community is that the relations and interactions described by Mihaly Csikszentmihalyi’s Systems Model of Creativity (SMoC) and the opportunity to influence these interactions by the usage of Stimuli of Invention (SI) are accelerating the processes of learning and education. The Maker Community itself offers the play area for young people and young at heard adults to live and foster their curiosity, and stimulating young people and their adults to be inventive and innovative in the long run

An engineered IN-1 Fab fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity

The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/κ antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 Fab fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 Fab fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 μM. The affinity of the unmutated IN-1 Fab fragment was 8-fold lower. The engineered Fab fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered Fab fragment as a reagent for promoting axonal regeneration in viv

Structure of the human heterodimeric transporter 4F2hc-LAT2 in complex with Anticalin, an alternative binding protein for applications in single-particle cryo-EM.

Cryo-EM structure determination of relatively small and flexible membrane proteins at high resolution is challenging. Increasing the size and structural features by binding of high affinity proteins to the biomolecular target allows for better particle alignment and may result in structural models of higher resolution and quality. Anticalins are alternative binding proteins to antibodies, which are based on the lipocalin scaffold and show potential for theranostic applications. The human heterodimeric amino acid transporter 4F2hc-LAT2 is a membrane protein complex that mediates transport of certain amino acids and derivatives thereof across the plasma membrane. Here, we present and discuss the cryo-EM structure of human 4F2hc-LAT2 in complex with the anticalin D11vs at 3.2 Å resolution. Relative high local map resolution (2.8-3.0 Å) in the LAT2 substrate binding site together with molecular dynamics simulations indicated the presence of fixed water molecules potentially involved in shaping and stabilizing this region. Finally, the presented work expands the application portfolio of anticalins and widens the toolset of binding proteins to promote high-resolution structure solution by single-particle cryo-EM

An Evolutionary Perspective of the Lipocalin Protein Family

The protein family of Lipocalins is ubiquitously present throughout the tree of life, with the exception of the phylum Archaea. Phylogenetic relationships of chordate Lipocalins have been proposed in the past based on protein sequence similarities, but their highly divergent primary structures and a shortage of experimental annotations in genome projects have precluded a well-supported hypothesis for their evolution. In this work we propose a novel topology for the phylogenetic tree of chordate Lipocalins, inferred from multiple amino acid sequence alignments. Sixteen jawed vertebrates with fair coverage by genomic sequencing were compared. The selected species span an evolutionary range of ∼400 million years, allowing for a balanced representation of all major vertebrate clades. A consensus phylogenetic tree is proposed following a comparison of sequence-based maximum-likelihood trees and protein structure dendrograms. This new phylogeny suggests an APOD-like common ancestor in early chordates, which gave rise, via whole-genome or tandem duplications, to the six Lipocalins currently present in fish (APOD, RBP4, PTGDS, AMBP, C8G, and APOM). Further gene duplications of APOM and PTGDS resulted in the altogether 15 Lipocalins found in contemporary mammals. Insights into the functional impact of relevant amino acid residues in early diverging Lipocalins are also discussed. These results should foster the experimental exploration of novel functions alongside the identification of new members of the Lipocalin family.España Ministerio de Ciencia e Innovación grant PID2019-110911RB-I0

Improved tetracycline repressors for gene silencing in mycobacteria

Tetracycline repressor (TetR)-controlled expression systems have recently been developed for mycobacteria and proven useful for the construction of conditional knockdown mutants and their analysis in vitro and during infections. However, even though these systems allowed tight regulation of some mycobacterial genes, they only showed limited or no phenotypic regulation for others. By adapting their codon usage to that of the Mycobacterium tuberculosis genome, we created tetR genes that mediate up to ∼50-fold better repression of reporter gene activities in Mycobacterium smegmatis and Mycobacterium bovis BCG. In addition to these repressors, for which anhydrotetracycline (atc) functions as an inducer of gene expression, we used codon-usage adaption and structure-based design to develop improved reverse TetRs, for which atc functions as a corepressor. The previously described reverse repressor TetR only functioned when expressed from a strong promoter on a multicopy plasmid. The new reverse TetRs silence target genes more efficiently and allowed complete phenotypic silencing of M. smegmatis secA1 with chromosomally integrated tetR genes

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochimica et Biophysica Acta - Biomembranes 1788 (2009): 1255-1262, doi:10.1016/j.bbamem.2008.12.016.Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na+/Ca2+ exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far- UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α- helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.The work was supported by Grants from the US National Science Foundation [MCB 0444598], Fondo Nacional para Investigaciones Científicas y Tecnológicas [PICT-05- 12397 and PICT-05-38073], Consejo Nacional de Investigfaciones Científicas y Técnicas [PIP 5118 and PIP 5593] Secretaría de Ciencia y Técnica Universidad Nacional de Córdoba, Argentina, Fondo Nacional para Ciencia y Técnica [S1-9900009046 and G- 2001000637] and Fundación Polar, Venezuela and The Rhode Island Idea Network of Biomedical Research Excellence (INBRE)