A high energy structure change in hemoglobin studied by difference hydrogen exchange

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Hemoglobin's allosteric transition involves significant conformational changes.
• Hydrogen exchange (HX) is a sensitive probe of protein dynamics and stability.
• Understanding these dynamics is crucial for hemoglobin function.

Purpose of the Study:

• To investigate the hydrogen exchange behavior of an allosterically responsive proton set in hemoglobin A.
• To correlate changes in hydrogen exchange rates with hemoglobin's allosteric state and cooperativity.
• To examine the impact of chemical modifications on these dynamic properties.

Main Methods:

• Studied hydrogen exchange rates in purified hemoglobin A and chemically modified variants.
• Utilized the local unfolding model of hydrogen exchange to interpret results.
• Performed experiments at pH 7.4 and 0 degrees C, with and without allosteric effectors like pyrophosphate and inositol hexaphosphate.
• Investigated modifications including iodoacetamide, N-ethylmaleimide, and carboxypeptidases A and B.

Main Results:

• A specific set of 5-6 protons exhibited large changes in exchange rates between oxyhemoglobin (20s half-time) and deoxyhemoglobin.
• In deoxyhemoglobin, this set split into slower (3 protons, 26h half-time) and faster (2-3 protons, ~2h half-time) components.
• Allosteric effectors (pyrophosphate, inositol hexaphosphate) further slowed the exchange of the slower protons (136h half-time).
• Chemical modifications generally accelerated the exchange of the slower protons, with factors correlating to changes in cooperativity.

Conclusions:

• The studied proton set is highly sensitive to hemoglobin's allosteric state, reflecting substantial changes in local free energy.
• The local unfolding model effectively explains the observed hydrogen exchange behavior.
• Chemical modifications that alter cooperativity also impact the dynamics of this specific hydrogen exchange set.