2025 Hyper Recent •CC0 1.0 Universal

This work is dedicated to the public domain. No rights reserved.

Access Preprint From Server
September 2nd, 2025
Version: 1
Brigham Young University
biochemistry
bioRxiv

Optimal TELSAM-Target Protein Linker Character is TargetProtein-Dependent

Pedroza Romo, M. J.Open in Google Scholar•Moody, J. D.Open in Google Scholar•Keliiliki, A.Open in Google Scholar•Averett, J.Open in Google Scholar•Gonzalez, J.Open in Google Scholar•Noakes, E.Open in Google Scholar•Wilson, E.Open in Google Scholar•Smith, C.Open in Google Scholar•Averett, B.Open in Google Scholar•Hansen, D.Open in Google Scholaret al.

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), semiflexible (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 semiflexible 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.

Similar Papers

bioRxiv
Wed Sep 03 2025
Enzymatic Function of an Intrinsically Disordered Protein
Intrinsically disordered proteins (IDPs) challenge the traditional structure-function paradigm by lacking a stable three-dimensional structure 1. While their roles as dynamic effectors, scaffolds, and molecular switches are well-established, it has been widely accepted that enzymatic activity requires a stably folded catalytic center 2. Here, we challenge this dogma by demonstrating that a 284-ami...
Lyalina, T.
•
Paim, L. M. G.
•
Bechstedt, S.
bioRxiv
Wed Sep 03 2025
Diffusive and advective fluid flow shapes chemoautotrophic bacterial communities and sulfur mineralogy in hydrothermal sediments off Milos
Hydrothermal fluid flow not only shapes mineral deposition on the ocean floor but also creates ecological niches by altering temperature and energy availability. In these niches, microbial life thrives and has an additional, often unrecognized impact on mineral formation. In a newly discovered vent field in medium depths off Milos, Greece, we show how contrasting hydrothermal regimes host fundamen...
Maak, J. M.
•
Röttgen, C.
•
Winkelhues, B.
•
Anagnostou, E.
...•
Elvert, M.
bioRxiv
Wed Sep 03 2025
Purified Zymogens Reveal Mechanisms of Snake Venom Metalloproteinase Auto-Activation
Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the ...
Hall, S.
•
Cardoso, I. A.
•
Wilkinson, M. C.
•
Carretero, M. M.
...•
Schaffitzel, C.
bioRxiv
Wed Sep 03 2025
Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging
Fluorescent proteins have transformed biological imaging, yet their limited photostability and brightness restrict their applications. We used deep learning-based de novo protein design methods to design binders to three bright, stable and cell-permeable dyes spanning the imaging spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). We obtain highly specific dye-binding proteins with lo...
Tran, L.
•
Sharma, S.
•
Klein, S.
•
Jurgens, D.
...•
Baker, D.
bioRxiv
Wed Sep 03 2025
Structural basis of QueC-family protein function in qatABCD anti-phage defense
QueC proteins are nucleoside biosynthesis enzymes required for production of the 7-deazaguanine derivative queuosine. Recently, QueC-family proteins were also shown to catalyze a deazaguanylation protein-nucleobase conjugation reaction in type IV CBASS bacterial anti-phage defense. Here we determine the structural basis of QueC-family protein function in a distinct bacterial immunity system named ...
Gao, A.
•
Wassarman, D. R.
•
Kranzusch, P. J.
bioRxiv
Wed Sep 03 2025
The anti-phage mechanism of a widespread trypsin-MBL module
Protease mediate activation of immune effectors is a conserved mechanism across life. This study identified a widespread and co-evolving trypsin-MBL module as a core effector in diverse bacterial immune systems (e.g., Hachiman, Argonaute, AVAST), protecting host via abortive infection. Focusing on Hachiman-trypsin-MBL, we demonstrated that, the protease activity of trypsin-HamAB is inhibited in th...
Huang, P.
•
Liu, J.
•
Guo, L. G.
•
Xu, D.
...•
Chen, M.
bioRxiv
Wed Sep 03 2025
Novel single-domain AA12 pyrroloquinoline quinone-dependent oxidoreductases directly and co-operatively drive lytic polysaccharide monooxygenase activity with AA8 module
The single-domain auxiliary activity family 12 (AA12) pyrroloquinoline quinone-dependent oxidoreductases and free AA8 modules are prevalent in cellulolytic fungi, however, their function in polysaccharide biodegradation is still confused. Here, we characterized three single-domain AA12 oxidoreductases and one free AA8 module from Thermothelomyces thermophilus and Thermothielavioides terrestris. Al...
Sun, Y.
•
Yuan, C.
•
Long, L.
•
Ding, S.
bioRxiv
Wed Sep 03 2025
The MLLT3 YEATS domain is a dual reader of histone marks (H3K9/18/27ac/cr) and ncRNA (7SK), linking epigenetic and RNA signaling to regulate hematopoiesis
The protein MLLT3 (AF9) is a critical regulator of hematopoiesis. The N-terminal YEATS domain of MLLT3 is an epigenetic reader that binds to acetylated as well as crotonylated lysine. Using PAR-CLIP, biochemical assays, and NMR based mapping of binding, we demonstrate that the YEATS domain of MLLT3 binds to a specific stem-loop region of the noncoding RNA 7SK. 7SK is a noncoding RNA with a well-do...
Boulton, A.
•
Kabra, A.
•
Achille, N.
•
Adelman, E. R.
...•
Bushweller, J.
bioRxiv
Tue Sep 02 2025
SH2-mediated steric occlusion of the C2 domain regulates autoinhibition of SHIP1 inositol 5-phosphatase
The Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase 1 (SHIP1) is an immune cell specific enzyme that regulates phosphatidylinositol-(3,4,5)-trisphosphate signaling at the plasma membrane following receptor activation. SHIP1 plays an important role in processes such as directed cell migration, endocytosis, and cortical membrane oscillations. Alterations in SHIP1 expressi...
Drew, E. E.
•
Nyvall, H. G.
•
Parson, M. A. H.
•
Talus, R. K.
...•
Hansen, S. D.
bioRxiv
Tue Sep 02 2025
Sticky salts: overbinding of monovalent cations to phosphorylations in all-atom forcefields
Phosphorylation is a major post-translational modification, which is involved in the regulation of the dynamics and function of Intrinsically Disordered Proteins (IDPs). We recently characterized a phenomenon, which we termed n-Phosphate collaborations (nP-collabs), where bulk cations form stable bridges between several phosphoresidues in all-atom molecular dynamic simulations. nP-collabs were fou...
Marien, J.
•
Puyo-Fourtine, J.
•
Prevost, C.
•
Sacquin-Mora, S.
•
Duboue-Dijon, E.