Expanding the single-molecule biochemistry toolkit
Prof. Dina Grohmann, Universität Regensburg
Single-molecule measurements have become an integral part of biological research. Among others, fluorescence-based approaches provide intriguing insights into the conformational, functional and structural diversity of biological molecules. However, the power of fluorescence-based single-molecule techniques, especially single-molecule FRET, cannot be fully exploited so far as it is a very time-consuming technique that requires the site-specific engineering of fluorescent reporters into the molecule of interest. The labelling and purification procedures strip a biomolecule of its natural binding partners and in many cases of its activity. Moreover, these sample preparation schemes are seldom applicable to cytoplasmic mammalian proteins.
In order to overcome this fundamental limitation of fluorescence-based single-molecule studies, we combined the biorthogonal labelling of proteins with organic dyes via unnatural amino acids and the single-molecule pulldown technique into a single workflow that allows for fast site-specific fluorescent labelling of proteins in prokaryotic and eukaryotic cellular extracts and subsequent single-molecule analysis of bimolecular complexes under near physiological conditions. This way, each and every cellular protein or protein complex can be directly pulled out of a cell lysate in a dye-labelled form carrying all native posttranslational modifications and can be detected and observed on the single-molecule level without the need for large-scale purification and further processing. This ultimately unleashes the complete prokaryotic and eukaryotic proteome for fast single-molecule fluorescence assays. In this talk I will discuss the newly developed sample preparation schemes and illustrate how it enabled us to investigate fragile proteins and protein complexes of the RNA-induced silencing (RISC) machinery on the single-molecule level.