Optimization of experimental procedure for assessing transition metal ion FRET in LeuT

Neurotransmitter sodium symporters (NSSs) are important for the regulation of neurotransmitters, such as dopamine and serotonin, which are involved in addiction and depression, along with several other diseases. The bacterial NSS, LeuT, has been proven to be a good model protein for the mammalian NSSs. Conformational changes of LeuT can be examined using transition metal ion Fo ̈rster Energy Resonance Transfer (tmFRET), where the energy transfer between a fluorescien dye and a di-histidine bound Ni2+ is measured. When utilizing tmFRET, free Ni2+ is added to the solution, which also adds to the unspecific quenching signal detected. It is therefore necessary to remove the signal from the unbound Ni2+, in order to properly investigate the impact of specific bound Ni2+. Here we investigate whether the effect of Zn2+ can be used to inhibit FRET contribution from specifically bound Ni2+ and thereby isolate the non-specific signal. We found that Zn2+ can competitively inhibit the binding of Ni2+ to the di-histidine motif of LeuT, thereby representing an easier, and perhaps more consistent, method for removing the signal from unbound Ni2+ during tmFRET measurements

Toxin inhibition in <i>C. crescentus</i> VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo

Ny viden om gamle ærtesorter

Organic RDD-projektet PEAS & LOVE undersøger, hvordan man kan at udnytte det store potentiale i gamle ærtesorter til dansk produktion af modne ærter til konsum

En veloverstået dyrkningssæson med gamle ærtelinjer i organic RDD-projektet Peas & Love

Projektet Peas & Love’s første opformering af gamle ærtelinjer er nu ved at være veloverstået. Af de i alt 300 gamle linjer inkluderet i projektet, blev 110 ærtelinjer udvalgt på baggrund af proteinindhold, udbytte og slægtskab. De udvalgte ærtelinjer blev dyrket på en økologisk mark, hvor de blev opformeret i kvadrater eller lange rækker alt efter mængden af ærtefrø i beholdningen. For nogle ærtelinjer var der kun ganske få ærter tilgængelige, og de blev derfor opformeret i drivhusets beskyttende rammer. I marken blev 13 moderne ærtesorter dyrket med de gamle linjer for at kunne sammenligne vækst, udbytte og smag under samme vækstforhold