Averett, J. | Search bioRxiv

bioRxiv2025

Optimal TELSAM-Target Protein Linker Character is TargetProtein-Dependent

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

biochemistry↗

bioRxiv2023

Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits

TELSAM crystallization promises to become a revolutionary tool for the facile crystallization of proteins. TELSAM can increase the rate of crystallization and form crystals at low protein concentrations without direct contact between TELSAM polymers and, in some cases, with very minimal crystal contacts overall (Nawarathnage et al., 2022). To further understand and characterize TELSAM-mediated crystallization, we sought to understand the requirements for the composition of the linker between TELSAM and the fused target protein. We evaluated four different linkers Ala-Ala, Ala-Val, Thr-Val, and Thr-Thr, between 1TEL and the human CMG2 vWa domain. We compared the number of successful crystallization conditions, the number of crystals, the average and best diffraction resolution, and the refinement parameters for the above constructs. We also tested the effect of the fusion protein SUMO on crystallization. We discovered that rigidification of the linker improved diffraction resolution, likely by decreasing the number of possible orientations of the vWa domains in the crystal, and that omitting the SUMO domain from the construct also improved the diffraction resolution. SynopsisWe demonstrate that the TELSAM protein crystallization chaperone can enable facile protein crystallization and high-resolution structure determination. We provide evidence to support the use of short but flexible linkers between TELSAM and the protein of interest and to support the avoidance of cleavable purification tags in TELSAM-fusion constructs.

biochemistry↗