Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Area of Science:

• Structural Biology
• Biochemistry
• Virology

Background:

• Cryo-electron microscopy (cryo-EM) has advanced significantly, enabling detailed imaging of macromolecules.
• Specimen-related challenges remain a focus for cryo-EM advancements.
• Low-molecular weight proteins present unique analytical difficulties.

Purpose of the Study:

• To develop a microchip-based toolkit for complementary structural and biochemical analysis of low-molecular weight proteins.
• To investigate the SARS-CoV-2 nucleocapsid (N) protein as a model system.
• To guide future pandemic preparedness through immuno-engineering strategies.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) for structural analysis of the SARS-CoV-2 N protein monomer.
• Engineered microchip-based immunoprecipitation assays for biochemical analysis.
• Performed molecular modeling of various coronavirus proteins.

Main Results:

• Determined cryo-EM structures of the N protein monomer, revealing a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain.
• Discovered the first antibody binding site on the N protein using microchip-based assays.
• Facilitated molecular modeling of pandemic and common cold-related coronavirus proteins.

Conclusions:

• The developed microchip toolkit enables comprehensive analysis of low-molecular weight proteins.
• Structural and biochemical insights into the SARS-CoV-2 N protein can inform therapeutic development.
• Findings support immuno-engineering strategies for enhanced pandemic preparedness.