Inhibition of ice recrystallization with designed twistless helical repeat proteins

Area of Science:

• Protein engineering and design
• Biophysics of ice interactions
• Crystallography

Background:

• Crystalline ice has a repetitive structure, mirrored by repeating motifs in natural ice-binding proteins (IBPs).
• Existing IBPs often feature beta-roll structures, limiting exploration of alternative protein scaffolds.

Purpose of the Study:

• To design and characterize a novel family of de novo ice-binding proteins with an alpha-helical structure.
• To investigate the structure-activity relationships of these engineered proteins in ice binding and ice recrystallization inhibition (IRI).

Main Methods:

• De novo protein design of ice-binding twistless alpha-helical repeat (iTHR) proteins.
• X-ray crystallography to determine protein structures.
• Site-specific mutagenesis and variation of repeat numbers to assess ice-binding activity and IRI.

Main Results:

• Successful design of iTHR proteins with a unique alpha-helical, planar layer structure.
• Demonstrated high solubility, thermostability, and ability to modulate ice crystal morphology.
• Achieved significant IRI activity comparable to native IBPs, with robust ice-binding activity across chemical variations.
• X-ray structures confirmed precise threonine orientation for ice surface complementarity.

Conclusions:

• The iTHR protein family represents a novel scaffold for ice-binding and IRI.
• These proteins provide a versatile platform for systematic studies of protein-ice interactions.
• The findings highlight the potential of de novo design for creating functional biomolecules with tailored ice-binding properties.