A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Human hemoglobin (HbA) exhibits cooperative oxygen binding, a complex process involving structural transitions.
• Modifying specific sites, like beta-93 sulfhydryl groups, can reveal insights into these transitions.
• Spin labels and NMR probes are valuable tools for studying protein conformation and dynamics.

Purpose of the Study:

• To investigate the binding of oxygen and a spin-labeled triphosphate to HbA modified at the beta-93 sulfhydryl groups.
• To model HbA with specific beta-chain modifications using spin-labeled triphosphate.
• To test the applicability of the concerted transition model (GCT model) to explain cooperativity and structural changes in modified HbA.

Main Methods:

• Covalent labeling of human adult hemoglobin (HbA) at beta-93 sulfhydryl groups with specific iodoacetamide derivatives.
• Studying the binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (SLTP) to modified HbA.
• Utilizing electron paramagnetic resonance (EPR) spectroscopy with spin labels and 19F NMR with trifluoracetone as probes.

Main Results:

• Oxygen and SLTP binding data for HbA modified with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (HbA-I) supported the GCT model.
• The GCT model successfully accounted for the environments sensed by spin labels and 19F NMR probes at the beta-93 sulfhydryl groups.
• The findings indicate that beta-93 probes detect quaternary (T to R) and tertiary structural changes upon ligand binding.

Conclusions:

• The concerted transition model (GCT model) effectively explains cooperativity in HbA, considering subunit nonequivalence.
• Conformation-sensitive probes at beta-93 reflect significant structural rearrangements during hemoglobin's functional cycle.
• Ligand binding to beta-heme induces tertiary changes at the alpha1-beta2 contact, consistent with the GCT model.