The His-tag as a decoy modulating preferred orientation in cryoEM
Raquel Bromberg1,2, Kai Cai1, Yirui Guo2
1Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX, United States.
Frontiers in Molecular Biosciences
|November 3, 2022
Summary
The His-tag influences protein behavior at the air-water interface during cryo-electron microscopy (cryoEM) grid preparation. Tagging strategies can be rationally designed to control these interactions for better structural studies.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Histidine tags (His-tags) are crucial for recombinant protein purification via affinity chromatography.
- Understanding protein behavior at interfaces is vital for structural and functional studies, particularly in cryo-electron microscopy (cryoEM).
Purpose of the Study:
- To investigate the impact of His-tag presence on the interaction of coproheme decarboxylase with the air-water interface during cryoEM grid preparation.
- To analyze how His-tagging affects protein orientation patterns and air-water interface interactions.
Main Methods:
- Comparative analysis of protein orientation in cryoEM grids with and without His-tag.
- Examination of particle orientations to infer protein-interface interactions.
Main Results:
- His-tag presence significantly alters preferred orientation patterns of coproheme decarboxylase at the air-water interface.
- The His-tag may mask native hydrophobic and hydrophilic patches, while its linker enhances alternative interface interactions.
Conclusions:
- His-tagging can modulate protein interactions with the air-water interface, influencing cryoEM sample preparation.
- Rational design of affinity tags and linkers presents a strategy to control protein behavior at interfaces for improved structural biology applications.
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