Design of single-layer beta-sheets without a hydrophobic core

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• The hydrophobic effect is a primary driver of protein folding, typically forming a hydrophobic core essential for structure.
• Outer surface protein A (OspA) has a stable, solvent-exposed beta-sheet lacking a hydrophobic core.

Purpose of the Study:

• To engineer larger, stable single-layer beta-sheets in OspA.
• To investigate the role of non-core interactions in protein structure stabilization.

Main Methods:

• Engineering OspA variants with extended beta-sheets by duplicating beta-hairpin units.
• Utilizing Nuclear Magnetic Resonance (NMR) and Small-Angle X-ray Scattering (SAXS) for structural analysis.
• Employing amide hydrogen-deuterium exchange and chemical denaturation to assess stability.

Main Results:

• Successfully created OspA variants with five- and seven-stranded beta-sheets as designed.
• Confirmed the structural integrity and stability of these extended beta-sheets.
• Demonstrated that intra- and inter-unit interactions, not a hydrophobic core, stabilize the beta-sheet structure.

Conclusions:

• Single-layer beta-sheet structures can be stabilized by internal interactions without a hydrophobic core.
• This finding expands the understanding of protein folding, misfolding, and design principles.
• Highlights alternative strategies for protein stabilization and engineering.