Melanie Wagner

Synthesis and application of fluorescent ras proteins for live-cell imaging.

Semisynthetic Ras proteins are efficient probes for cell‐biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid‐phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N‐terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

Bridging the gap between cell biology and organic chemistry: chemical synthesis and biological application of lipidated peptides and proteins

Abstract. We have developed a basic concept for studying cell biological phenomena using an interdisciplinary approach starting from organic chemistry. Based on structural information available for a given biological phenomenon, unsolved chemical problems are identified. For their solution, new synthetic pathways and methods are developed, which reflect the state of the art in synthesising lipidated peptide conjugates. These compounds are used as molecular probes for the investigation of biological phenomena that involve both the determination of biophysical properties and cell biological studies. The interplay between organic synthesis, biophysics and cell biology in the study of protein lipidation may open up new and alternative opportunities to gain knowledge about the biological phenomenon that could not be obtained by employing biological techniques alone. This fruitful combination is highlighted using the Ras protein as an outstanding example. Included herein is: the development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of the biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and proteins in cell biological experiments.

The chemical biology of Ras lipidation.

Abstract In this review, we summarize the successful interplay between three disciplines, organic synthesis, biophysics and cell biology, in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in molecular detail. Highlighting the example of the Ras proteins, we show how the development of new synthetic methodologies paved the road to the synthesis of lipidated peptides and by a combination of chemical and molecular biological techniques lipidated proteins as molecular tools. We further give an overview of the results of the biophysical properties and biological activities of the molecules synthesized by means of this interdisciplinary approach. This successful combination of different disciplines led to a better understanding of the selective targeting of Ras and related lipoproteins to the plasma membrane.

Generation and Characterization of Ras Lipoproteins Based on Chemical Coupling

Chemically synthesized truncated Ras proteins are coupled to C-terminal Ras peptides via a maleimidocaproyl linker. The resulting product is isolated by extraction with Triton X-114. The biological activity of these oncogenic Ras lipoproteins can be determined in a cell-based differentiation assay by microinjection into PC12 cells.

Long-term Care Provision and the Well-Being of Spousal Caregivers: An Analysis of 138 European Regions

Abstract Objectives The individual burden of caring for one’s relatives not only depends on care characteristics but is also related to contextual factors. The objective of this study is to determine whether regional formal long-term care provision is linked to the well-being of spousal caregivers introducing the concept of “control” as central pathway to explain this link. Method We applied multilevel analysis using data from the Survey of Health, Ageing and Retirement in Europe (SHARE) from over 29,000 Europeans and 1,800 spousal caregivers located in 138 regions in 11 countries to analyze the effects of regional contexts on caregiver well-being. The provision of formal care in a region was measured by the number of long-term care beds in nursing and residential homes among the older population. Results We found that spousal caregivers’ well-being, measured in terms of life satisfaction, loneliness, and depression, was positively linked to the regional availability of formal care, which is partly due to higher perceived control in regions with more formal care provision. Discussion Our results suggest that formal care supply is essential not only for care recipients but also for caregivers: perceived alternatives to the private care arrangement go along with greater well-being of informal caregivers.