Biology subjects

Olasz, B.

Publications and source records attributed to Olasz, B..

2 recordsLinked to original sources

bioRxiv2023

Fixed-target time-resolved crystallography at XFELs: the scourge of light contamination but reduced sample consumption

X-ray free electron laser (XFEL) light sources have allowed for the rapid growth of time-resolved structural experiments, which provide crucial information on the function of biological machines and their mechanisms. We set out to commission the SwissMX fixed-target sample delivery system at the SwissFEL Cristallina experimental station using the PSI developed MISP-chip for pump-probe time-resolved experiments. To characterise the system, we used the light-sensitive protein crystals of the Light-Oxygen-Voltage domain 1 (LOV1) from Chlamydomonas reinhardtii. Using different experimental settings, the adjacent-well light contamination was carefully assessed, indicating that it is crucial to control the light scattering from solid supports otherwise significant contamination can occur. However, our results show that, after the initial experiments and parameter refinement, the opaque MISP-chips are suitable for pump-probing a light-sensitive protein. This crystallographic experiment also probed the sub-millisecond structural dynamics of the LOV1 and indicated that at {Delta}t=10 s the covalent thioether bond is already established between the reactive Cys57 and FMN cofactor. This experiment validated the crystals to be suitable for in-depth follow up studies of the still poorly understood signal transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared to the most successful system used at XFEL, the high-viscosity extruder. This development creates the prospect of an exciting increase in XFEL project throughput for the field.

biophysics↗

bioRxiv2023

Structural analysis of the Sterile alpha motif (SAM) domain of the Arabidopsis mitochondrial tRNA import receptor

Mitochondria are membrane bound organelles of endosymbiotic origin with limited protein coding capacity. As a consequence, the continual import of nuclear-encoded protein and nucleic acids such as DNA and small non-coding RNA is required and essential for maintaining organelle mass, number and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with 6 SAM domains per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of Tric1 SAM domain was essential for protein function whereby mutation of Gly241 resulted in the disruption of the oligomer and the loss of RNA binding capability in Tric1. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype suggesting the oligomerization is essential for function in planta. AlphaFold2 structure prediction of the SAM domain and Tric1 support a cyclic hexamer generating a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane. Our results highlight the importance of oligomerization of Tric1 for protein function.

biochemistry↗