Allosteric intermediates indicate R2 is the liganded hemoglobin end state

Area of Science:

• Protein structure and dynamics
• Biochemistry and molecular biology
• Allosteric regulation mechanisms

Background:

• Hemoglobin allostery is a classic model for protein conformational changes.
• Understanding hemoglobin's quaternary structure transitions is crucial for deciphering its function.
• Previous models proposed T-R and R-R2 allosteric pathways.

Purpose of the Study:

• To elucidate the allosteric pathway of hemoglobin using chemically crosslinked variants.
• To investigate the structural basis of hemoglobin's conformational states.
• To assess the physiological relevance of the R2 conformation.

Main Methods:

• X-ray crystallography was used to determine the structures of two chemically crosslinked, fully liganded hemoglobins.
• High-resolution structural data (2.3 A and 2.6 A) were obtained for alpha2beta82CA82beta and alpha2beta82ND82beta hemoglobins.
• Comparative structural analysis was performed to identify intermediate conformations.

Main Results:

• The crosslinked hemoglobins adopted intermediate conformations distinct from the canonical R and T states.
• These intermediate structures were found to lie between the R and R2 states, not R and T.
• The observed conformations provide direct structural evidence for the R2 state's role.

Conclusions:

• The study supports an allosteric pathway involving T -> R -> R2 transitions in hemoglobin.
• The findings underscore the physiological importance of the R2 conformation in hemoglobin function.
• This work refines our understanding of protein allostery using hemoglobin as a model system.