Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Stromal interaction molecule 1 (STIM1) acts as an endoplasmic reticulum (ER) Ca(2+) sensor.
• STIM1 activation initiates store-operated Ca(2+) entry, a critical signaling pathway in diverse cell types.
• Understanding STIM1 protein domains is essential for its functional and structural studies.

Purpose of the Study:

• To identify functional and stable domains of human STIM1 (hSTIM1) for large-scale recombinant production.
• To characterize recombinant hSTIM1 constructs for in vitro biochemical and biophysical applications.

Main Methods:

• Cloning of approximately 200 hSTIM1 constructs with variations in residues, length, and transmembrane domain presence.
• Expression and purification analyses of cloned constructs.
• Functional and integrity verification using surface plasmon resonance, nuclear magnetic resonance, and thermostability assays.

Main Results:

• Nearly 50% of hSTIM1 constructs were successfully expressed and purified with high quality.
• 25% of the high-quality constructs included the integral membrane domain.
• Verified functionality and integrity of selected hSTIM1 domains through biophysical techniques.

Conclusions:

• Identified stable and well-behaved domains of the hSTIM1 protein.
• These domains are suitable for large-scale production and future in vitro biochemical and biophysical studies.
• Facilitates further research into STIM1-mediated calcium signaling.