Abstract

TELSAM crystallization can be 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. To further understand and characterize TELSAM crystallization, we sought to understand the requirements for the composition of the linker confirmation between TELSAM and the fused target protein. We evaluated four linkers, AlaAla, AlaVal, ThrVal, and ThrThr, 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 can improve diffraction resolution, likely by decreasing the possible orientations of the vWa domains in the crystal and that omitting the SUMO domain from the construct also improves the diffraction resolution.The hydrogenase enzyme is involved in the formation and degradation of hydrogen in various biochemical reactions in multiple organisms. It represents a promising avenue for biohydrogen production in the future. The hydrogenase uses a unique iron-sulfur cluster (termed the H-cluster) to generate hydrogen. Three maturase enzymes: HydF (~46 KDa), HydE (~40KDa), and HydG (~55KDa), are involved in the H-cluster assembly/maturation process. Structural characterization of complexes of the maturase enzymes is lacking and needed to better understand the H-cluster assembly. We have expressed and purified the hydrogenase maturase complex. Sizeexclusion chromatography supports the existence of a ~175 KDa complex containing dimeric HydF and monomeric HydG and HydE. We have initial negative stain 2D class averages and a low-resolution 3D reconstruction of the maturation complex. Our next step will be to determine a high-resolution cryo-EM structure of the maturase complex.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2023-02-15

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd12663

Keywords

TELSAM, Crystallization, Hydrogenase Maturase complex, Cryo-EM

Language

english

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