Biology subjects

Gensch, T.

Publications and source records attributed to Gensch, T..

4 recordsLinked to original sources

bioRxiv2021

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

The growth rate {micro} of bacteria depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for a few bacterial species, in particular Escherichia coli, but are missing for most bacterial phyla. In this study, we systematically analysed the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower {micro}max. Ribosomes were quantified via single-molecule localization microscopy (SMLM) using fluorescently tagged ribosomal proteins and via RNA/protein ratio. Both methods revealed a non-linear relationship with little change in ribosome abundance below {micro} = 0.4 h-1 and a steep increase at higher {micro}. Unlike E. coli, C. glutamicum keeps a large pool of active ribosomes at low {micro}, but the translation elongation rate declines from [~]9 amino acids s-1 at {micro}max to <2 aa s-1 at {micro} < 0.1 h-1. A model-based approach shows that depletion of translation precursors at low growth rates can explain the observed decrease in translation elongation rate. Nutrient up-shift experiments support the hypothesis that maintenance of excess ribosomes during poor nutrient conditions enables C. glutamicum to quickly restart growth when conditions improve.

microbiology↗

bioRxiv2021

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.

biophysics↗

bioRxiv2020

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Forster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptors constitutive activity. For the agonist-bound A2AAR, we detected faster (390{+/-}80 s) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

biophysics↗

bioRxiv2020

Accessing mitochondrial protein import in living cells by protein microinjection

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

cell biology↗