Disulfide-constrained Fabs overcome target size limitation for high-resolution single particle cryoEM

Area of Science:

• Structural Biology
• Biochemistry
• Biophysics

Background:

• High-resolution protein structures are crucial for biological understanding and drug development.
• Single-particle cryo-electron microscopy (cryoEM) excels for large complexes but struggles with small proteins (<50 kDa) due to low signal-to-noise and alignment challenges.
• Existing methods using scaffold proteins to increase target size often introduce flexibility, hindering high-resolution structure determination.

Purpose of the Study:

• To develop a novel strategy for determining high-resolution structures of small proteins using cryoEM.
• To engineer antibody fragments (Fabs) into conformationally stable entities suitable for cryoEM analysis of small targets.
• To demonstrate the efficacy of this approach for resolving biologically relevant small proteins.

Main Methods:

• Iterative engineering of antibody fragments (Fabs) to create conformationally Rigid Fabs.
• Introduction of strategic disulfide bonds within Fabs to enhance structural rigidity and stability.
• Application of Rigid Fabs in single-particle cryoEM for structure determination of small proteins.

Main Results:

• Successfully transformed Fabs into Rigid Fabs, characterized by enhanced conformational stability.
• Achieved high-resolution cryoEM structures (2.3–2.5 Å) for two small proteins: Angiopoietin-2 (Ang2, 26 kDa) and KRAS (21 kDa).
• Demonstrated the general applicability of Rigid Fabs across different species, frameworks, and chimeric constructs.

Conclusions:

• The disulfide-constrained Rigid Fab strategy provides a robust solution for overcoming the size limitations of small proteins in cryoEM.
• This method enables high-resolution structural studies of previously challenging small, disease-related proteins.
• Offers a versatile approach for advancing structural biology and drug discovery for small protein targets.