Challenges in the interpretation of protein h/d exchange data: a molecular dynamics simulation perspective

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Hydrogen/deuterium exchange (HDX) is a key technique for protein structure analysis.
• Amide protection in HDX is traditionally attributed to intramolecular H-bonding and burial of NH sites.
• Recent findings challenge these fundamental assumptions of HDX.

Purpose of the Study:

• To investigate the factors governing "open" (exchange-competent) and "closed" (exchange-incompetent) amide environments in proteins.
• To explore the dynamic features of protein structure and solvation using molecular dynamics (MD) simulations.
• To re-evaluate the predictive power of H-bonding and solvent accessibility for HDX protection.

Main Methods:

• Utilized all-atom molecular dynamics (MD) simulations to dynamically assess protein structure and solvent interactions.
• Analyzed hydrogen bonding (backbone and side chain) and solvent accessibility for NH sites in ubiquitin.
• Investigated the role of crystallographic water and water network dynamics in amide protection.

Main Results:

• MD simulations explained HDX properties for 57 out of 72 NH sites based on H-bonding and solvent accessibility.
• A significant number of exposed NH sites showed unexpected protection, while others with similar features were unprotected.
• H-bonding to crystal water and restricted water dynamics did not account for the anomalous protection observed in surface amides.

Conclusions:

• Standard criteria for HDX protection (H-bonding, burial) are insufficient to explain observed protection patterns.
• Current models may lead to misleading interpretations of protein structure derived from HDX data.
• Further quantum mechanical studies are likely necessary to fully understand HDX rate determinants, despite computational challenges.