Position-dependent effects of loop proline residues on Fab stability and aggregation

Area of Science:

• Protein Engineering
• Biochemistry
• Immunology

Background:

• Proline residues are crucial for protein structural stability due to conformational constraints.
• The specific role of proline in the stability and properties of antibody fragments is not fully understood.

Purpose of the Study:

• To investigate the structural role of proline residues in the loop regions of adalimumab's antigen-binding fragment (Fab).
• To evaluate the impact of native proline substitutions and proline introductions on Fab stability and function.

Main Methods:

• Site-directed mutagenesis to substitute native prolines with glycine (e.g., H:P41G, H:P14G).
• Engineering proline variants in the constant domain based on sequence conservation.
• Circular dichroism spectroscopy, antigen binding analysis, and differential scanning calorimetry to assess structural and thermal properties.
• Evaluation of aggregation under pH-shifted stress conditions.

Main Results:

• Loop-region proline substitutions (H:P41G, H:P14G) did not significantly alter secondary structure or antigen binding.
• Differential scanning calorimetry revealed position-dependent effects on thermal stability, with H:P41G showing decreased melting temperature and H:P14G indicating disrupted domain unfolding.
• A proline-introduced variant (H:K209P) demonstrated partial stabilization and reduced aggregation under stress.
• Native proline residues contribute to Fab stability and interdomain cooperation.

Conclusions:

• Loop-region proline residues are important for the structural stability and interdomain cooperation of antibody fragments.
• Antibody engineering strategies involving proline introduction require careful consideration of the surrounding structural context.
• Proline engineering can be a viable approach to enhance the stability and reduce aggregation of antibody-based therapeutics.