Authors: Pedroza Romo, Maria Jose
Source: ScholarsArchive  (Brigham Young University) (article, 2025-08-22)
Field: Materials Science — Materials Chemistry

Engineering Protein Linkers for Optimized TELSAM-DARPin Fusion and Crystallization With Electric Field-Based Polymer Orientation Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukemia protein (TELSAM) to a protein of interest significantly enhances crystallization and diffraction quality. TELSAM is a pH-dependent, polymer-forming crystallization chaperone that, when covalently fused to a protein, forms a well-ordered crystal lattice. However, the success of this method depends on two factors: 1) the design of the linker connecting TELSAM to the target protein, which influences both crystal packing and target orientation, and 2) the prevention of TELSAM polymer flipping, a phenomenon that disrupts lattice periodicity and impairs structure determination. To address the first challenge, we performed a systematic study to evaluate how different linker types between TELSAM and the protein of interest affect crystal formation and quality. We designed multiple constructs with rigid (helical fusion), semi-flexible (Pro-Alan), and flexible (poly-Gly) linkers of varying lengths to fuse a designed ankyrin repeat protein (DARPin) to TELSAM. We generated semi-flexible and flexible constructs with and without a 10xHis tag based on previous studies that revealed the 10xHis tag can either facilitate or hinder the ordered crystallization of target proteins. We found that short semi-flexible and rigid linkers yield high-quality crystals within 24 hours with a DARPin target protein, but that flexible linkers perform best with a TNK1-UBA domain target protein. Our data show the use of a short yet flexible or semi-flexible linker between TELSAM and the target protein facilitates protein crystallization and high-resolution structure determination (Chapter 2). We also investigated "polymer flipping," where TELSAM polymers adopt alternating N-to-C orientations, disrupting lattice periodicity. In a CMG2 vWa fusion construct (SUMO-1TEL-Thr-Val-CMG2 vWa), polymer flipping complicated structure determination despite good diffraction. To prevent this, we applied an electric field during crystallization using a custom capacitor device to align TELSAM polymer dipoles. The field improved resolution and reduced mosaicity in HEWL crystals. However, in the SUMO-1TEL-Thr-Val-CMG2 vWa construct, it accelerated crystallization but induced partial cleavage of the target, resulting in heterogeneous crystals with only 37% indexed reflections. These findings demonstrate that electric fields can enhance lattice order and reduce disorder, but their effects may be construct-dependent (Chapter 3). Together these studies contribute to advancing the understanding of TELSAM fusion protein crystallization and offer potential solutions to challenges faced in high-resolution structural determination. Linker ; Crystallization ; Protein crystallization ; Polymer ; Covalent bond ; Fusion protein ; Fusion ; Crystallization of polymers ; Protein domain Enzyme Structure and Function ; Force Microscopy Techniques and Applications ; Heat shock proteins research