Identification of an allosterically sensitive unfolding unit in hemoglobin

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Allosteric regulation in proteins like hemoglobin involves conformational changes affecting function.
• Understanding the molecular mechanisms of these changes is crucial for deciphering protein behavior.
• Existing models often link hydrogen-exchange rates to solvent accessibility, but this may not fully capture dynamic processes.

Purpose of the Study:

• To investigate the dynamic behavior of specific amide protons in the hemoglobin beta chain during allosteric transitions.
• To test the validity of penetration-dependent models for hydrogen exchange in proteins.
• To explore the contribution of protein segment dynamics to allosteric free energy.

Main Methods:

• Utilized hydrogen-exchange studies to monitor amide proton dynamics in hemoglobin beta chains.
• Employed a functional labeling technique with tritium to selectively label exchange-sensitive sites.
• Applied a protein fragment separation method to locate labeled segments and quantify exchange rates.

Main Results:

• Identified seven allosterically sensitive amide NH protons in the F-FG helical segment of the hemoglobin beta chain.
• Observed that these protons exchange at similar rates regardless of their surface or internal location and move in unison during allosteric form changes.
• Demonstrated a 30-fold faster exchange rate in liganded hemoglobin compared to the deoxy form, inconsistent with simple penetration-dependent models.

Conclusions:

• Results support a local unfolding model where protein fluctuations make protons accessible in a transient state, rather than simple solvent penetration.
• The F-FG helical segment is destabilized in oxyhemoglobin, contributing approximately 2 kcal to the allosteric free energy, favoring the deoxy state.
• The developed functional labeling method is effective for identifying protein segments involved in functional interactions.