Optimal TELSAM-Target Protein Linker Character is TargetProtein-Dependent

Pedroza Romo, M. J.; Moody, J. D.; Keliiliki, A.; Averett, J.; Gonzalez, J.; Noakes, E.; Wilson, E.; Smith, C.; Averett, B.; Hansen, D.; Nickles, R.; Bradford, M.; Soleimani, S.; Smith, T.; Nawarathnage, S.; Samarawickrama, P.; Kelsch, A.; Bunn, D.; Abiodun, W.; Tsubaki, E.; Doukov, T.; Brown, S.; Stewart, C.

doi:10.1101/2025.08.29.672704

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bioRxiv · 10.1101/2025.08.29.672704

Optimal TELSAM-Target Protein Linker Character is TargetProtein-Dependent

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Abstract

Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukaemia protein (TELSAM) to a protein of interest has been shown to significantly enhance crystallization propensity. TELSAM is a pH-dependent, polymer-forming protein crystallization chaperone which, when covalently fused to a protein of interest, forms a stable, well-ordered crystal lattice. However, despite its success, a challenge persists in that crystal quality and diffraction limits appear to be heavily dependent on the choice of linker between TELSAM and the protein of interest, with identification of a functional linker relying on trial-and-error methods. Likewise, previous studies revealed that the 10xHis tag at the TELSAM N-terminus can either facilitate or hinder the ordered crystallization of target proteins attached via flexible or semi-flexible linkers. To address these challenges, we designed multiple constructs with several types of linkers--rigid (helical fusion), semi-flexible (Pro-Alan), and flexible (poly-Gly)--of varying lengths to fuse a designed ankyrin repeat protein (DARPin) to the TELSAM C-terminus. Semi-flexible and flexible linker constructs were made with and without the 10xHis tag. Our findings indicate that short semi-flexible and rigid linkers consistently yield large crystals within 24 hours with a DARPin target protein, but that flexible linkers perform best with a TNK1 UBA domain target protein. Removing the 10xHis tag enhanced crystallization rates, improved crystal morphology, and increased the crystallization propensity of semi-flexible and flexible linker constructs. While removing the His tag did not have a significant effect on crystal size, it improved the diffraction limits and crystal quality of the 1TEL-PA-DARPin construct. These results suggest that the ideal linker selection primarily depends on the properties of the target protein. Our data support the recommendation to use a short yet flexible or semi-flexible linker between TELSAM and the target protein to facilitate protein crystallization and high-resolution structure determination. SynopsisIn this study, we examine the effect of short to medium-length flexible, semi-flexible, and rigid linkers on the crystallization of a DARPin fused to the 1TEL protein crystallization chaperone, demonstrating that while rigid linkers impair crystallization and reduce diffraction quality, the ideal linker character remain target-protein dependent. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672704v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@b90d1org.highwire.dtl.DTLVardef@1508e7dorg.highwire.dtl.DTLVardef@1baf0d1org.highwire.dtl.DTLVardef@1843df_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pedroza Romo, M. J., Moody, J. D., Keliiliki, A., Averett, J., Gonzalez, J., Noakes, E., Wilson, E., Smith, C., Averett, B., Hansen, D., Nickles, R., Bradford, M., Soleimani, S., Smith, T., Nawarathnage, S., Samarawickrama, P., Kelsch, A., Bunn, D., Abiodun, W., Tsubaki, E., Doukov, T., Brown, S., Stewart, C.. 2025-09-02. Optimal TELSAM-Target Protein Linker Character is TargetProtein-Dependent. https://doi.org/10.1101/2025.08.29.672704

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