Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity

Area of Science:

• Protein structure and stability
• Enzyme catalysis and engineering
• Molecular biology and genetics

Background:

• Interhelical turns are crucial for protein structure.
• Previous studies focused on simpler protein models.
• Assessing turn tolerance in complex proteins requires sensitive methods.

Purpose of the Study:

• To evaluate sequence substitution tolerance in a larger, complex protein's interhelical turn.
• To investigate the influence of secondary structure and tertiary interactions on substitution patterns.
• To leverage in vivo selection for catalytic activity to assess functional tolerance.

Main Methods:

• Randomization of solvent-exposed and buried residues within an interhelical turn.
• Utilizing in vivo selection for catalytic activity in Escherichia coli chorismate mutase.
• Analyzing substitution patterns to identify residue preferences and constraints.

Main Results:

• >63% of tripeptides could functionally replace native turn residues, even with altered backbone conformations.
• Solvent-exposed positions showed a preference for hydrophilic residues.
• Buried turn residues exhibited a strict requirement for hydrophobic aliphatic amino acids, unlike helical residues.

Conclusions:

• Tertiary interactions impose strong constraints on interhelical turn substitutions, while secondary structure has a weaker influence.
• Protein design strategies should consider the distinct roles of secondary structure and tertiary interactions in constraining sequence.
• This study provides insights into the adaptability and constraints of protein structural motifs.