Biology subjects

Heimerl, T.

Publications and source records attributed to Heimerl, T..

6 recordsLinked to original sources

bioRxiv2025

Mechanistic insight into the oligomerization of Arabidopsis CRY1 and its inhibition by BIC1

Cryptochromes (CRY) convert blue light signals into biological responses, however, the molecular processes underlying their activation are not fully understood. In this study, we uncover the molecular mechanism underlying the blue-light activation of Arabidopsis CRY1 using time-resolved native mass spectrometry combined with kinetic modelling - an approach that allows us to monitor light-driven complex formation with temporal and molecular resolution. We show that CRY1 activation follows a defined, reversible assembly pathway in which monomers rapidly form dimers that then assemble into tetramers. A quantitative two-step model captures the dynamic interplay between light-driven assembly and thermal disassembly. Strikingly, ATP accelerates tetramer formation and stabilises oligomers by tuning the underlying photochemistry of the FAD chromophore. In contrast, the Blue-light Inhibitor of Cryptochromes 1 (BIC1) acts as a potent antagonist. We found that BIC1 binds to CRY1 even in the dark, with a significant increase in binding strength under blue-light conditions. BIC1 not only blocks CRY1 oligomerisation, but also actively dismantles pre-assembled tetramers. This disassembly process is light-independent and occurs regardless of CRY1s redox state. Together, these findings reveal a finely balanced regulatory system in which ATP and BIC1 act as opposing regulators to control CRY1 activation. This work provides a kinetic and mechanistic framework for reversible cryptochrome signalling and highlights how blue light responses can be precisely modulated at the molecular level.

plant biology↗

bioRxiv2024

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

evolutionary biology↗

bioRxiv2022

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown. Here, we characterize the cell-free production of infectious bacteriophage T7 virions under simulated microgravity. Rotation in a 2D-clinostat increased the number of infectious particles compared to static controls. Quantitative analyses by mass spectrometry, immuno-dot-blot and real-time PCR showed no significant differences in protein and DNA contents, suggesting enhanced self-assembly of T7 phages in simulated microgravity. While the effects of genuine space conditions on the cell-free synthesis and assembly of bacteriophages remain to be investigated, our findings support the vision of a cell-free synthesis-enabled "astropharmacy".

synthetic biology↗

bioRxiv2022

Selective accumulation of matrix proteins inside of peroxisomal subdomains

Formation of specialized reaction spaces prevents interference between distinct cellular pathways. Peroxisomes are cellular compartments involved in a large diversity of metabolic processes. How peroxisomes differentiate into subpopulations and by which mechanism intraorganellar domains are formed remains largely elusive. Here, we report on enzymes from the fungus Ustilago maydis, which accumulate inside of peroxisomal subdomains. We describe a short peptide motif (Thr-Ile-Ile-Val) sufficient to trigger focal localization. Mining for proteins with similar motifs uncovered several peroxisomal matrix proteins that accumulate in intraorganellar foci. These foci are enriched in the enzyme urate oxidase - a typical constituent of the paracrystalline core of peroxisomes. Upon peroxisome proliferation uneven distribution of focal structures results in the formation of peroxisome subpopulations with different protein content. The underlying principle of subdomain formation is evolutionary conserved in mammalian peroxisomes and formation of similar foci was also observed inside of mitochondria. We propose that peroxisomal proteins show an individual propensity to self-assemble. This formation of protein aggregates appears to be a ubiquitous driving force to spatially organize the peroxisomal proteome.

cell biology↗

bioRxiv2022

ATPase activity of B. subtilis RecA affects the dynamic formation of RecA filaments at DNA double strand breaks

RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed "threads", which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, and still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduces formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. While lowered ATP-binding activity only moderately affected RecA dynamics, loss of ATPase activity lead to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and non-specifically DNA-bound molecules into filaments upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Our data suggest that RecA/ssDNA filaments change in subcellular localization and length involving ATP-driven homology search.

microbiology↗

bioRxiv2019

Characterization of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) with flagella are an ancestral eukaryotic trait that has been lost in several lineages, for example in flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) strains to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute strain to identify a set of genes associated with moss male infertility. Defects in mammal and algal homologs of these genes coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. As a proof of principle, we generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Indeed, the Ppccdc39 mutant resembles the male infertile Gransden strain phenotype. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of P. patens Gransden, causing regulatory differences of e.g. the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility. We propose that P. patens spermatozoids might be employed as an easily accessible system to study male infertility of human and animals.

plant biology↗