Structural difference between two ATP-binding sites of heavy meromyosin revealed by the dynamic fluorescence...

Area of Science:

• Biochemistry
• Molecular Biology
• Protein-ligand interactions

Background:

• Heavy meromyosin (HMM) is a key motor protein involved in muscle contraction.
• Understanding ATP binding site characteristics is crucial for elucidating HMM's enzymatic mechanism.
• 1,N6-ethenoadenylyl imidodiphosphate (e-AMPPNP) is a fluorescent ATP analog used to probe binding sites.

Purpose of the Study:

• To investigate the binding characteristics of e-AMPPNP to the ATP binding sites of heavy meromyosin.
• To determine if the two ATP binding sites on HMM exhibit differential ligand binding.
• To assess the accessibility of the bound e-AMPPNP's fluorescent group to solvent.

Main Methods:

• Acrylamide fluorescence quenching was employed to study e-AMPPNP binding to HMM.
• Varying molar ratios of HMM to e-AMPPNP were used to analyze binding behavior.
• Stern-Volmer plots were analyzed to determine binding constants and ligand accessibility.

Main Results:

• Modified Stern-Volmer plots indicated non-linear quenching, suggesting distinct binding environments.
• Two Stern-Volmer constants (0.89 M⁻¹ and 13 M⁻¹) were obtained for bound e-AMPPNP.
• Analysis revealed that approximately 94% of bound e-AMPPNP exhibited a lower affinity (0.89 M⁻¹), while 6% showed higher affinity (13 M⁻¹).
• Under different conditions, 50% of bound e-AMPPNP showed higher affinity (13 M⁻¹).
• The fluorescent group of e-AMPPNP bound to the high-affinity site was significantly more isolated from the solvent.

Conclusions:

• Heavy meromyosin possesses two distinct ATP binding sites with differential affinities for e-AMPPNP.
• One ATP binding site exhibits significantly higher affinity and sterically hinders the fluorescent probe.
• These findings provide insights into the heterogeneity of ATP binding sites in HMM and their functional implications.