High-pH NMR to Identify Macromolecular Hydrogen-Bonds and Foldons

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Hydrogen bonds are crucial for protein structure determination using NMR.
• Identifying hydrogen bonds is challenging for marginally stable proteins due to insufficient protection from deuterium exchange.
• Traditional deuterium exchange methods have limitations in accuracy and applicability.

Purpose of the Study:

• To explore high-pH NMR as an alternative method for identifying backbone amide protons involved in hydrogen bonds.
• To assess the accuracy and applicability of high-pH NMR for mapping hydrogen bonds in proteins.
• To investigate protein unfolding hierarchies and dynamics under alkaline conditions.

Main Methods:

• Utilized NMR spectroscopy (2D 1H-15N HSQC and TOCSY) at high pH (10-11) to detect persistent amide protons.
• Analyzed approximately 750 amide sites across ten proteins with known structures.
• Performed alkaline unfolding experiments on selected proteins to observe signal attenuation and identify stable structural elements ('foldons').

Main Results:

• High-pH NMR identified hydrogen bonds with approximately 91% accuracy, exceeding the ~80% accuracy of traditional deuterium exchange.
• The method revealed an unfolding hierarchy in proteins, with 'foldons' representing regions with the most persistent hydrogen bonds at high pH.
• Foldon characteristics correlated with structural features like inter-residue contact density in beta-sheets and helical propensity in coiled coils.

Conclusions:

• High-pH NMR is a sensitive, fast, inexpensive, and broadly applicable technique for mapping hydrogen bonds in marginally stable or partially folded proteins.
• This approach offers new insights into protein dynamics and unfolding pathways under alkaline conditions.
• The findings provide a valuable alternative for structural studies where traditional methods are insufficient.