We determined the structure of the human Thirty-eight-negative kinase-1 Ubiquitin-Associated domain (TNK1 UBA) and the proposed binding modes of UBA to TNK1. UBA was fused to TELSAM, a protein crystallization chaperone capable of forming a 6 member helical polymer that forces a target protein (UBA in this case) to crystallize with it. We cloned, expressed and purified the TELSAM-UBA construct (1TU). We did a crystallization trial with commercial and custom-made crystallization solutions. After obtaining crystals we harvested the crystals and used the Synchrotron from SSRL to diffract 1TU crystals. The diffraction data was refined using crystallization software. We were able to solve the previously unknown TNK1 UBA structure by using the already known TELSAM structure to solve the X-ray phases and then solving the for UBA attached to the TELSAM. We found that the flexible linker connecting TELSAM to UBA could only be extended a little ways and that various crystals of a single 1TEL-target construct could have slightly different crystal packing. In addition, we found that the TELSAM constructs were able to crystallize at significantly low protein concentrations (0.1 mg/mL) and that the target proteins fused to TELSAM maintained similar conformations at different concentrations. We also observed significantly fewer crystals at high Mg-formate concentrations, which we hypothesize is due to Mg<sup>2+</sup> ions blocking TELSAM polymerization by attaching to TELSAM-TELSAM binding sites. We also observed that 1TU constructs did have higher crystallographic b-factors (uncertainty in atomic positions) at the UBA C-terminus. Additionally, we were able to use the solved UBA structure to computationally model and experimentally test potential binding modes of UBA to TNK1. In conclusion, we solved a previously unknown protein structure (TNK1 UBA) using the TELSAM crystallization chaperone and determined some of the favorable TELSAM construct crystallization conditions.

TELSAM protein chaperone helps determine the TNK1 UBA domain structure and favorable TELSAM crystallization conditions

Where thousands of chemistry professionals meet to share ideas and advance scientific and technical knowledge. Spring 2023 abstracts, posters, and presentations will launch at the end of March.

ACS Spring 2023

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.