Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.
| Autor: | Gajjar PL, Romo MJP, Litchfield CM, Callahan M, Redd N, Nawarathnage S, Soleimani S, Averett J, Wilson E, Lewis A, Stewart C, Tseng YJ, Doukov T, Lebedev A, Moody JD |
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| Jazyk: | angličtina |
| Zdroj: | BioRxiv : the preprint server for biology [bioRxiv] 2023 May 15. Date of Electronic Publication: 2023 May 15. |
| DOI: | 10.1101/2023.05.12.540586 |
| Abstrakt: | 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. Synopsis: We 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. |
| Databáze: | MEDLINE |
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