Of bits and bugs

Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins

Assessing Information Waste in Lean Product Development

Lean Product Development seeks to enhance the efficiency of product development projects by reducing and eliminating non-value-adding activities or waste, which can exist on every process level. The value stream through product development processes is a flow of information, and hence waste exists in interpersonal communication. The study elaborates the hypothesis that most information transfers do not add value to the product. It was further theorized that different means of communication are better suited for different kinds of information, at least from the lean point of view. In order to understand the occurrence and ramifications of waste in product development information flows, the information transferred between team members was analyzed in two student product development projects. With the help of a paper-based value stream map, frequencies of waste drivers in information, the share of waste in information transfers, the interdependencies of waste and means of communication, as well as timeliness of information transfers were analyzed. The study’s results show that waste is omnipresent in product development information transfers, as only twelve percent of all information transfers contribute value to the product, and nearly half of the information transfers could have been omitted without a decrease in product value. Assuming that preparing, sending, receiving and retrieving information accounts for most of the time spent in product development processes, an enormous theoretical potential for efficiency enhancements could thus be identified

Information and Communication in Lean Product Development

In this thesis, the implications and influences that information and communication impose on lean product development in general, as well as the development of a lean Product Development Value Stream Display (lean PDVSD) in specific, are discussed theoretically, studied and analyzed. First, the concepts of information and communication are discussed theoretically from a lean perspective. Definitions are provided and aspects of importance to lean processes are deduced. Furthermore, requirements for an envisioned lean PDVSD are gathered systematically. Existing Value Stream Mapping tools are introduced and shortly discussed, and the current development of the envisioned lean PDVSD is briefly reported. A preliminary, paper-based version is provided

Crystal structure, nucleic-acid binding properties, and dimerization model of Pur-alpha

This study characterizes Pur-α structurally and functionally. Pur-α is a highly conserved RNA- and DNA-binding protein involved in a multitude of cellular processes such as transcription, replication, cell cycle control, and mRNA transport. No homologous proteins with known structures are available. X-ray crystallography is often hampered by the lack of diffraction-quality protein crystals. This study demonstrates how this bottleneck was overcome by the combination of iterative use of sensitive bioinformatics tools and structure determination of a bacterial homolog. The identification of three repeat regions (PUR repeats) in eukaryotic Pur-α enabled the detection of a bacterial homolog, which corresponds to one PUR repeat. The crystal structure of Borrelia burgdorferi Pur-α was solved at 1.9 Å and was employed for precise domain boundary prediction for the Drosophila melanogaster ortholog. Therewith it became possible to obtain diffraction-quality crystals of eukaryotic Pur-α. The crystal structure of D. melanogaster Pur-α repeats I-II was solved at 2.1 Å and shares a highly conserved fold with B. burgdorferi Pur-α. One PUR repeat has an overall ββββα− topology, and two PUR repeats interact with each other to form a globular PUR domain. Small angle X-ray scattering (SAXS) analysis together with analytical size-exclusion chromatography provided evidence that dimerization of full length Pur-α requires PUR repeat III. PUR repeat III is proposed to form a PUR domain with a PUR repeat III from another Pur-α molecule. Surface envelopes calculated from SAXS data comply with this dimerization model. DNA- as well as RNA-binding properties of Pur-α were examined by filter binding assays and electrophoretic mobility shift assays. Structure-guided mutagenesis identified the β-sheets of the PUR domain as the nucleic-acid binding surface. To assess the protein-binding properties of D. melanogaster Pur-α, a yeast-two-hybrid screen was commissioned and evaluated. It confirmed the self-interaction of Pur-α and yielded Arrestin1, LaminC, Eye and Cka as putative previously unknown interaction partners