Hydrogen bonding and biological specificity analysed by protein engineering

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Hydrogen bonds play a crucial role in molecular recognition and biological specificity.
• Enzyme-substrate interactions are fundamental to biological processes.
• Tyrosyl-tRNA synthetase is a key enzyme in protein synthesis.

Purpose of the Study:

• To investigate the quantitative contribution of complementary hydrogen bonding to biological specificity.
• To determine the impact of unpaired hydrogen bond donors/acceptors on enzyme-substrate binding energy.
• To elucidate the role of charge in hydrogen bonding interactions within enzyme active sites.

Main Methods:

• Site-directed mutagenesis was used to engineer the tyrosyl-tRNA synthetase.
• Deletion of specific amino acid side chains to alter hydrogen bonding potential.
• Measurement of binding energy changes between the engineered enzyme and its substrate.

Main Results:

• Deletion of an uncharged hydrogen-bond donor or acceptor resulted in a modest decrease in binding energy (0.5-1.5 kcal mol-1).
• The presence of an unpaired, charged hydrogen-bond donor or acceptor significantly weakened binding energy by approximately 3 kcal mol-1.
• This indicates that charged hydrogen bonds contribute substantially more to binding energy than uncharged ones.

Conclusions:

• Complementary hydrogen bonding is a critical determinant of biological specificity.
• The energetic contribution of hydrogen bonds is significantly influenced by their charge state.
• Protein engineering provides a powerful tool to dissect the energetic contributions of specific molecular interactions.