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Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139 344) with TBK1.
The shorter construct results in a signal of higher SNR and slower folding/unfolding kinetics, thereby facilitating the detection of otherwise fast structural transitions.
However, a slightly shorter construct (535 700) yielded crystals in a tetragonal space group that proved amenable to structure determination.
The protease resistant fragment of ∼25 kDa that was lost in ΔF508 CFTR did arise in constructs truncated behind NBD1 or downstream, but not in the shorter construct truncated behind MSD1 (data not shown), implying that this fragment related to NBD1.
For Myosin XI-6, the "nocoil" construct was expressed rather than the full tail region, because for unknown reasons, the shorter construct produced more consistent labeling.
We identified that while the full-length promoter activity increased by 1.8-fold on TLX overexpression, there was a negligible effect on the shorter construct.
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One interesting exception was a very short construct (amino acids 735 759) that corresponds precisely the linear bipartite NLS peptide motif alone, the minimal set of amino acids for importin binding [27].
The shortest construct (−1768/+38Luc) displayed a low but yet detectable activity in endothelial cells, corresponding to a 93% loss of that in −8409/+38Luc but still with an endothelial/fibroblast ratio of 3.4.
In addition, transcription activity in shortest construct (−245 ESR1 Luc).
This may explain the drop in activity observed in the shortest construct.
In the current series we used a short construct design in order to reduce the stress on the terminal screws.
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