Design and synthesis of a globin fold

Area of Science:

• Protein engineering and computational biology
• Structural biology and biophysics

Background:

• Designing de novo proteins with specific structures and functions is a significant challenge in biochemistry.
• Understanding the relationship between amino acid sequence, protein folding, and biological function is crucial for protein design.

Purpose of the Study:

• To develop and validate a computational method for designing an amino acid sequence that folds into a specific globular protein structure.
• To create a synthetic myoglobin variant (DG1) with an artificial sequence that mimics the structure and heme-binding properties of natural myoglobin.

Main Methods:

• Utilized a knowledge-based 3D-1D compatibility function and recursive protein 3D profile generation for sequence design.
• Incorporated specific constraints for heme-binding site residues (His64, His93) and employed a repulsive function to avoid steric clashes.
• Synthesized the designed gene, expressed the protein in Escherichia coli, and characterized its structure and function using biophysical techniques (SEC, CD, X-ray scattering, NMR).

Main Results:

• A novel 153-amino acid sequence (DG1) was designed, sharing 26% sequence identity with natural myoglobin.
• DG1 successfully folded into a monomeric, compact, globular, and highly helical structure in aqueous solution, similar to the target myoglobin structure.
• DG1 demonstrated heme binding with appropriate spectroscopic properties, although it did not stably bind molecular oxygen.

Conclusions:

• The developed computational method is effective for designing foldable protein sequences with desired structures.
• The study provides insights into sequence-structure-function relationships, highlighting the importance of sequence selection for achieving specific biological functions.
• Further research is needed to optimize side-chain conformational diversity for improved functional properties like oxygen binding.