Allosteric intermediates in hemoglobin. 1. Nanosecond time-resolved circular dichroism spectroscopy

Area of Science:

• Biophysics
• Structural Biology
• Protein Dynamics

Background:

• Hemoglobin (Hb) cooperativity involves allosteric transitions between R (relaxed) and T (tense) states.
• Understanding these transitions is crucial for deciphering oxygen transport regulation.
• Photolyzed hemoglobin-CO complex (HbCO) serves as a model system to study these dynamics.

Purpose of the Study:

• To investigate the real-time protein relaxations in Hb following CO photodissociation.
• To elucidate the mechanism of the R to T allosteric transition at room temperature.
• To characterize the structural changes at the alpha 1 beta 2 interface during this transition.

Main Methods:

• Time-resolved circular dichroism (TRCD) spectroscopy in the near-UV, UV, and Soret regions.
• Analysis of TRCD signals reflecting aromatic residue and porphyrin transitions.
• Integration of TRCD data with kinetic information from time-resolved absorption studies.

Main Results:

• TRCD in the near-UV region identified T-like structural changes at the alpha 1 beta 2 interface within nanoseconds.
• Porphyrin-based TRCD signals indicated modulation by tertiary and quaternary structural changes.
• The alpha 1 beta 2 interface shifted towards a T-like state significantly before the full equilibration of the T state deoxyhemoglobin.

Conclusions:

• The R to T allosteric transition in photolyzed HbCO is a stepwise process.
• Conformational changes propagate from the heme to the protein interface via the F helix.
• TRCD provides a sensitive probe for early structural events in allosteric transitions.